<figure><img src="image_68.png" /></figure>

FIGURE 8. Venn diagram representation for the character "presence of anterolateral processes on lachrymal" for which Macrourinae, *Squalogadus* and *Trachyrincus* taxa instantiate the state "present". Selected taxa (in red) are simply dragged from the taxa list and dropped in the main window.
<figure><img src="image_69.png" /></figure>

FIGURE 9. Venn diagram and tree representations for the character “presence of anterolateral processes on lacrymal”. Selected taxa (in red) are simply dragged from the taxa list and dropped in the main window.
<figure><img src="image_70.png" /><figcaption>**FIGURE 10.** Modification of *Squalogadus* position within the tree, the branch is dragged from one node and dropped to another.</figcaption></figure>

**Character analysis** – From the list of characters, LisBeth computes their three-item statements (3is) with their fractional weights. A text file (with the extension ".3iz") is automatically generated.

**Search** – LisBeth implements branch and bound algorithms, applied with the “Search for the compatibility trees” command (Fig. 11). The “Execute 3ia tool” windows specify defaults for the search options (Fig 12), e.g. “branch and bound”, “load the target file when finished”, etc. The information processing is detailed in the “LisBeth window” (Fig. 13), e.g. number of retained trees, elapsed time (in % of the search), etc... The optimal trees are stored in the file.3iz. They are listed in the interface “Optimal trees” window (Fig.14).
<figure><img src="image_71.png" /><figcaption>FIGURE 11. Search menu: search for compatible trees.</figcaption></figure>

<figure><img src="image_72.png" /><figcaption>FIGURE 12. Details of the Executive 3ia tool, main options are selected by default.</figcaption></figure>
<figure><img src="image_73.png" /><figcaption>FIGURE 13. Processing information details.</figcaption></figure>

**Summary tree** – LisBeth summarizes the optimal trees reconstructing a tree from the list of their common 3is, hence the name “intersection tree” for the summary tree (Cao *et al.* 2009). This function is given by the default options (Fig.12) (“secondary intersection tree”). With the interface, the intersection tree is always labeled [0] and can be displayed in the main window (Fig. 14).
<figure><img src="image_74.png" /><figcaption>FIGURE 14. Optimal tree window with, in red and labeled [0], the Intersection tree also displayed in the main window.</figcaption></figure>

**Save trees** – Each tree which is being displayed in the interface main window can be saved as a vector file format, using the “Save image” command from the “File” menu (Fig. 15). Several representations are available via the main window buttons, varying according to the branch relative length, the squared angle / oblique branches or the Venn diagram / rooted tree representation. Trees can be edited by dragging taxa to a given position and saved using the “Fix” button, Fig. 16.
<figure><img src="image_75.png" /><figcaption>**FIGURE 15.** File menu: Save image.</figcaption></figure>

<figure><img src="image_76.png" /><figcaption>**FIGURE 16.** Interface buttons specifying representation options e.g. the branch relative length, the squared angle / oblique branches or the Venn diagram / rooted tree representation.</figcaption></figure>

**Support** – LisBeth calculates various indexes: the per-character RI, the overall-RI and the completeness index (Compl., introduced in Zaragüeta Bagils *et al.* 2012). The overall-RI and Compl. are stored in the file.3iz and displayed in the interface main window (Fig. 14). The per-character-RI values are stored in a table (.3ri) and displayed in front of the listed characters under the “interpretation mode” (see below).

**Character history** – Character history can be inferred under the “interpretation mode” of the “Operation” menu (Fig. 17). It is displayed (Fig. 18) with coloured boxes listing the character states (e.g. 2:1 stands for state “1” of character “2”), “green” meaning unambiguous synapomorphy and “red” homoplasy. This information is available for the selected characters (e.g. the “Presence of anterolateral processes on lachrymal” character in Fig. 18; for all characters Fig. 19) or for a given node (Fig. 20). The relative placement of coloured boxes can be modified by dragging them to a given position. Under the “interpretation mode”, the “Display the tree” command (Fig. 21) allows for the character lookup in independent windows. Character states can be displayed as well using the “State” button.
<figure><img src="image_77.png" /><figcaption>FIGURE 17. Operation menu: Interpretation mode; display of an independent window where the « Source » contains the characters and the « Target » the tree(s).</figcaption></figure>
<figure><img src="image_78.png" /><figcaption>FIGURE 18. The evolution of selected character (in red in the Character window) is traced on the tree of the main window with a code-colour, here green for synapomorphy. For the sake of clarity, each box can be dragged in a given position without changing its link to the node.</figcaption></figure>
<figure><img src="image_79.png" /><figcaption>FIGURE 19. The evolution of all characters (in red in the Character window) is traced on the tree of the main window with a code-colour, here green for synapomorphy, red for homoplasy and orange if both synapomorphy and homoplasy are present for a single node. For the sake of clarity, each box can be dragged in a given position without changing its link to the node.</figcaption></figure>
<figure><img src="image_80.png" /><figcaption>FIGURE 20. Display of supporting synapomorphies for a given node with a code-colour, here green for synapomorphy. Each box can be dragged into a given position without changing its link to the node.</figcaption></figure>
<figure><img src="image_81.png" /><figcaption>FIGURE 21. Display of any character in an independent window.</figcaption></figure>

Interoperability with other programs and other functionalities

**Character export** – LisBeth exports 3is matrices (with 3is fractional weights) formatted for other phylogenetic programs (e.g. PAUP* 4b10, Swofford 2002; TNT, Goloboff *et al.* 2008). The “Export” command of LisBeth Toolbox allows for the inclusion of specific commands in the exported nexus or tnt file, e.g. name of the tree file, number of replicates (“replica”) etc...(Fig. 22).
<figure><img src="image_82.png" /><figcaption>**FIGURE 22.** Export to Nexus format. The options “hold”, “replica” etc… are available when exporting to TNT format.</figcaption></figure>

**Character import** – LisBeth imports characters from phylogenetic matrices and from Xper² (Ung *et al.* 2010) knowledge bases. Matrices must be enhanced with hierarchy of states for each character (Fig. 23) so that LisBeth can automatically reconstruct hierarchical characters and generate a text file (.3ia). Xper² program (Fig. 24) implements descriptions of taxa and distinguishes missing data (“unknown state”) from inapplicable data (thanks to parent-child relationships depending on the relevance of descriptors); hierarchical characters are automatically reconstructed from descriptors when specifying a hierarchy of states (Fig. 25).
<figure><img src="image_83.png" /><figcaption>FIGURE 23. Character state hierarchy enhanced matrix.</figcaption></figure>

<figure><img src="image_84.png" /><figcaption>FIGURE 24. Xper² « Edit items mode » screen-shot displaying descriptions of taxa.</figcaption></figure>
<figure><img src="image_85.png" /></figure>

**FIGURE 25.** Syntax for the import of phylogenetic characters from Xper² program. [1] stands for the character label. 2:1 stands for the state “1” of the second Xper² descriptor. A character is represented as a hierarchy of states. A single character (e.g. [3]) can include states from several Xper2 descriptors, e.g. 3rd and 4th) since hierarchy allows for the representation of inherited relationships.

**Tree import** – The “Import” command of LisBeth Toolbox imports trees from other phylogenetic programs, e.g. PAUP* 4b10 and TNT (Fig. 26).

<figure><img src="image_86.png" /></figure>

**FIGURE 26.** Import trees from PAUP* 4b10.
**Extract specific information** – The “Extract from .3iz” command of LisBeth Toolbox allows for the extraction of the selected information from a file (.3iz). The operation presented in Fig. 27 results in the extraction of the Intersection tree from the “gadiformes.3iz” file and its storage in a new file. The table with per-character RI values may also be updated in order to retain values for the intersection tree only.

<figure><img src="image_87.png" /><figcaption>**FIGURE 27.** Extract the Intersection tree from the 3iz file. The “Extraction .3ri” option is also selected: the table with per-character RI values will present values for the intersection tree only.</figcaption></figure>

**RI values** – The “RI” command of LisBeth Toolbox calculates the retention index values (Archie 1989; Farris 1989) for any pair of trees or {character, tree} (Fig. 28). This command is particularly helpful when trees have been imported from other programs.
<figure><img src="image_88.png" /><figcaption>FIGURE 28. Calculation of RI values from external list of characters or trees.</figcaption></figure>

LisBeth is the only software that implements hierarchical representation of characters for 3ia. It is a package of programs primarily written in C language for improved performance, including parallel computation when needed.

LisBeth has a user-friendly interface written in Python language (www.python.org).

LisBeth offers a ToolBox with new features providing more flexibility and advanced functionalities. Based on the same ergonomics, the LisBeth ToolBox has a user-friendly interface written in Python language too.

Requirements and availability:

Software name: LisBeth

Version: 1.3 (2013.01.27)

Software homepage:

http://lis-upmc.snv.jussieu.fr/lis/?q=en/resources/software/lisbeth

Operating system(s): Windows, Linux version under test.

Programming language: C (executables) and Python (interface)

Other requirements: Python (from 2.7 version) interpreter

License: This program is licensed under a Attribution-NonCommercial- NoDerivative Works 3.0 Unported (CC BY-NC-ND 3.0).

Cost: free.
References

Archie, J.W. (1989) *Homoplasy Excess Ratios: New Indices for Measuring Levels of Homoplasy in Phylogenetic Systematics* and a Critique of the Consistency Index. Systematic Biology, 38 (3), 253-269.

Cao, N., Zaragüeta Bagils, R. & Vignes-Lebbe, R. (2007) Hierarchical representation of Hypotheses of Homology. *Geodiversitas*, 29 (1), 5-15.

Cao, N., Bourdon, E., El Azawi, M. & Zaragüeta Bagils, R. (2009) Three-item analysis and parsimony, intersection tree and strict consensus a biogeographical example. *Bulletin de la Société géologique de France*, 180(1), 13-15.

Cohen, D.M., Inada, T., Iwamoto T., & Scialabba., N. (1990) FAO species catalogue. Vol. 10. Gadiform fishes of the world (Order Gadiformes). An annotated and illustrated catalogue of cods, hakes, grenadiers and other gadiform fishes known to date. FAO Fisheries Synopsis. No. 125, Vol. 10. FAO (Eds) Rome, 442 pp.

Corvez, A. (2012) *L'origine de la Mégaphylle chez les Monilophytes*. [PhD dissertation]. Muséum national d'Histoire naturelle, Paris (Eds) France, 276 pp.

Endo, H. (2002) Phylogeny of the Order Gadiformes (Teleostei, Paracanthopterygii). *Memoirs of the Graduate School of Fisheries Sciences*, 49(2), 75-149.

Escalante, T., Rodríguez, G., Cao, N., Ebach, M.C. & Morrone J.J. (2007) *Naturwissenschaften*, 94, 561-565.

Farris, J.S. (1989) The Retention Index and Homoplasy Excess. *Systematic Zoology*, 38(4), 406-407.

Farris, J.S. (1997) Cycles. *Cladistics*, 13, 131-144.

Farris, J.S. (2010) Systematic foundering. *Cladistics*, 26, 1-15.

Farris, J.S. (2012) 3ta Sleeps with the fishes. *Cladistics*, 28(4), 422-436.

Farris, J.S. & Kluge, A.G. (1998) The Brief History of Three-Taxon Analysis. *Cladistics*, 14, 349-362.

Gilbert, C.H. & Hubbs, C.L. (1916) Report on the Japanese macrouroid fishes collected by the United States Fisheries steamer Albatross in 1906, with a synopsis of the genera. *Proceedings of the United States National Museum*, 51(2149), 135-214.

Giorna, M.E. (1809) Mémoire sur des poissons d'espèces nouvelles et de genres nouveaux. Plus: Suite et conclusion du mémoire (pp. 177-180). *Mémoires de l'Académie Imperiale des Sciences, Littérature et Beaux-arts de Turin*, 9, 1-19, 177-180.

Goloboff, P.A., Farris J.S. & Nixon K.C. (2008) TNT, a free program for phylogenetic analysis. *Cladistics*, 24(5), 774-786.

Goode, G.B. & Bean, P.W. (1896) Oceanic ichthyology. A treatise on the deep-sea and pelagic fishes of the world, based chiefly upon the collections trade by the streamers BLADE, ALBATROSS and FISHHAWK in the northwestern Atlantic. *Memoirs of the Museum of Comparative Zoölogy at Harvard College*, 22, 1-553.

Günther, A.C.L.G. (1873) 1873, Andrew Garrett's fische der Südsee, beschrieben und redigirt von A.C.L.G. Günther. *Journal des Museum Godeffroy*, 2(3), 128.

Günther, A.C.L.G. (1878) Preliminary notices of deep-sea fishes collected during the voyage of H.M.S. 'Challenger'. *The Annals and Magazine of natural History*, (5)2(7), 17-28, 179-187, 248-251.
Günther, A.C.L.G. (1880) Report on the shore fishes procured during the voyage of H.M.S. 'Challenger' in the years 1873-1876. *Challenger Report (Zoology)*, 1(6), 1-82.

Harvey, A.W. (1992) Three-taxon statements: more precisely, an abuse of parsimony? *Cladistics*, 8, 345-354.

Jordan, D.S. & Evermann, B.W. (1898) The fishes of north and middle America: a descriptive catalogue of the species of fish - like vertebrates found in the waters of North America, north of the Isthmus of Panama. *Bulletin of the United States National Museum*, 47, 2183-3136.

Kitching, I.J., Foreyn P.L., Christophern J., Humphriesn J. & Williams, D.W. (1998) *Cladistics: the theory and practice of parsimony analysis*. The Oxford University Press (Eds) Oxford, 228 pp.

Kluge, A.G. (1994) Moving targets and shell games. *Cladistics*, 10, 403-413.

Ladiges, P.Y., Prober, S.M., & Nelson, G. (1992) Cladistic and biogeographic analysis of the 'blue ash' Eucalypts. *Cladistics*, 8, 103-124.

Ladiges, P.Y., McFadden, G.I., Middleton, N., Orlovich, D.A., Treloar, N. & Udovicic, F. (1999) Phylogeny of *Melaleuca, Callistemon*, and related genera of the *Beaufortia* suballiance (Myrtaceae) based on 5S and ITS-1 spacer regions of nrDNA. *Cladistics*, 15, 151-172.

Oken, L. (1817) V. Kl. Fische. *Isis* (Oken), 8(148), 1779-1782.

McCulloch, A.R. (1926) Report on some fishes obtained by the FIS 'Endeavour' on the coasts of Queensland, New South Wales, Victoria, Tasmania, South and South-western Australia. Part V. *In*: Dannevig, H.C. (Eds), *Biological results of the fishing experiments carried on by the FIS 'Endeavour'* pp. 157-216.

Moreau, C.E (1881) Histoire naturelle des poissons de la France. Masson G. (Eds) Paris.

Morrone, J.J. & Carpenter, J.M. (1994) In search of a method for cladistic biogeography: an empirical comparison of component analysis, Brooks parsimony analysis, and three-area statements. *Cladistics*, 10, 99-153.

Nelson, G. J. & Platnick, N.I. (1991) Three-taxon statements: A more precise use of parsimony? *Cladistics*, 4:351-366.

Nelson, G.J. & Ladiges, P.Y. (1992) Information Content and Fractional Weight of Three-Item Statements. *Systematic Biology*, 41(4), 490-494.

Nelson, G.J. & Ladiges, P.Y. (1993) Missing data and three-item analysis. *Cladistics*, 9, 111-113.

Nelson, J. (1994) *Fishes of the World. 3rd edition*. John Wiley & Sons (Eds), New York, 600 pp.

Nelson, J. (2006) *Fishes of the World. 4th edition*. Jhon Wiley & Sons (Eds) New Jersey, 601 pp.

Parenti, L.R. & Ebach, M.C. (2009). *Comparative Biogeography: Discovering and Classifying Biogeographical Patterns of a Dynamic Earth*. University of California Press (Eds) Berkeley, 312 pp.

Patterson, C. & Johnson, G.D. (1995) The intermuscular bones and ligaments of teleostean fishes. *Smithsonian Contributions in Zoology*, 559, 1-78.

Rafinesque, C.S. (1810a) *Indice d'ittiologia siciliana; ossia, catalogo metodico dei nomi latini, italiani e siciliani dei pesci, che si rinvengono in Sicilia disposti second un metodo natural e seguito da un'appendice che contiene la descrizione di alcuni nuovi pesci siciliani*. Presso Giovanni del Nobolo (Eds) Messina, 70 pp.

Rafinesque, C.S. (1810b) *Caratteri di alcuni nuovi generi e nuove specie di animali e piante della Sicilia, con varie osservazioni sopra i medisimi*. Sanfilippo (Eds) Palermo. 1-105.
Roa-Varón, A. & Ortí, G. (2009) Phylogenetic relationships among families of Gadiformes (Teleostei, Paracanthopterygii) based on nuclear and mitochondrial data. *Molecular Phylogenetics and Evolution*, 52, 688-704.

Swofford, D.L. (2002) *PAUP*: phylogenetic analysis using parsimony (*and other methods), version 4. Sunderland: Sinauer.

Thompson, W. (1940) On a new genus of fishes from India. *Charlesworth's Magazine of Natural History*, 4, 184-187.

Udovicic, F., McFadden, G. I. & Ladiges, P.Y. (1995) Phylogeny of *Eucalyptus* and *Angophora* based on 5S rDNA spacer sequence data. *Molecular Phylogenetics and Evolution*, 4, 247-256.

Ung, V., Dubus, G., Zaragüeta Bagils, R. & Vignes-Lebbe, R. (2010) Xper²: introducing e-taxonomy. *Bioinformatics*, 26(5), 703-704.

Wilkinson, M. (1994a) Three-taxon statements: When is a parsimony analysis also a clique analysis? *Cladistics*, 10, 221-223.

Wilkinson, M. (1994b) Common cladistic information and its consensus representation: reduced Adams and reduced cladistic consensus trees and profiles. *Systematic Biology*, 43(3), 343-368.

Williams, D.M. & Ebach, M.C. (2008) *Foundations of Systematics and Biogeography*. Springer (Eds) New York, 300 pp.

Zaragüeta Bagils, R. & Bourdon, E. (2007) Three-item analysis: Hierarchical representation and treatment of missing and inapplicable data. *Comptes Rendus Palevol*, 6, 527-534.

Zaragüeta Bagils, R., Ung, V., Grand, A., Vignes-Lebbe, R., Cao, N. & Ducasse, J. (2012) LisBeth: New cladistics for phylogenetics and biogeography. *Comptes Rendus Palevol*, 11(8), 563-566.
Annexe V : Comparative biogeography and
Geographic Speciation of North American highland
fishes from the Mesa Central to the Canadian Shield

Hoagstrom C. W., **Ung V.**, Taylor K. (2013)

*(*Journal of Biogeography*, **accepté**)
# Title: Miocene Rivers and Tertiary Taxon Cycles Clarify the Comparative Biogeography of North American Highland Fishes

Author names: Christopher W. Hoagstrom¹*, Visitheary Ung², Kathie Taylor³

Author research addresses: ¹Department of Zoology, Weber State University, Ogden, UT 84408, USA, ²CNRS UMR 7207 CR2P, 75005 Paris, France, ³Argenta Ecological Consultants, Salt Lake City, UT, 84171, USA.

*Correspondence: Chris Hoagstrom, Department of Zoology, Weber State University, 2505 University Circle, Ogden, UT, 84408-2505, USA.

E-mail: christopherhoagstrom@weber.edu

Running head: Comparative biogeography of highland fishes

Word count, Abstract (line 13) through References (line 829): 9762

## ABSTRACT

**Aim** To (1) use recently published phylogenies of six widely distributed clades of highland-type fishes in a comparative analysis that investigates relations among North American highlands, (2) construct a map of relevant (pre-historic) river geography, and (3) apply ecological paradigms to interpret patterns of highland-fish cladogenesis.

Principal questions are: (1) does highland endemism correspond to pre-historic river drainages, and (2) what ecological concept explains patterns of speciation?

**Location** Twenty-two North American highlands, east to west from Appalachia to the Basin and Range, north to south from the Canadian Shield to the Mesa Central.

**Methods** We (1) used three-item analysis to find shared highland-area relations, (2) used dated phylogenies and geological literature to construct a map of relevant, pre-historic river drainages, and (3) applied the taxon-cycle concept to interpret results.

**Results** Three-item analysis identified 369 most-parsimonious trees with a retention index of 80.4%. An intersection tree reconstructed from common three-area statements had a completeness index of 80.3%. Nodes on the tree identified seven major branches, of one to eight highland areas each, that were congruent with Late Miocene river drainage patterns. Sister-area relations within nodes were congruent with Plio-Pleistocene events. Older taxa have
restricted distributions and some younger members of each clade are broadly distributed, consistent with predictions
of the taxon cycle.

**Main conclusions** Progenitors of highland endemics were widespread across an aggraded, alluvial landscape by the Early Miocene. Middle Miocene drainage rearrangements facilitated further dispersal. Accelerated erosion in the Middle Miocene, caused by tectonic uplift and climate change, created highland-type (sediment-starved) habitats. Parallel colonization and adaptation to these habitats and geographical isolation in the Late Miocene and early Pliocene created highland endemics. Plio-Pleistocene drainage rearrangements allowed tolerant (derivative) taxa to disperse and again colonize highland areas, including emerging ones.

KEYWORDS

Endemism, inland aggradation, drainage rearrangement, Miocene, parallel evolution, preglacial rivers, reciprocal illumination, taxon cycle, tectonism, three-item analysis
## INTRODUCTION

Methods of Comparative Biogeography provide a means to test geological and phylogenetic hypotheses and develop new testable hypotheses that bring about "reciprocal illumination" (Rosen, 1978; Parenti & Ebach, 2009). Recent studies of North American freshwater fish distributions (Miller, 2005; Page & Burr, 2011) and phylogenetics (Table 1) along with recent studies of North American geomorphological history (Galloway et al., 2011) provide unprecedented opportunity for reciprocal illumination. Here, we incorporate this information into a comparative biogeographical study and combine biogeographical and geological evidence to construct the (to our knowledge) first map of Late Miocene rivers among North American highlands. The map and accompanying ecological interpretations are presented as tools for future hypothesis testing.

Major highland areas of North America (Fig. 1a) have long been recognized as centres of aquatic biodiversity. Highland areas share the geographic qualities of locally high elevation and topographic relief. Highland streams have locally high gradients, coarse substrates (i.e., gravel, cobble, bedrock), and cool waters. The insular nature of highland-type habitats corresponds to endemism of (for example) crayfishes (Crandall & Templeton, 1999), salamanders (Highton, 1995), and fishes (Mayden, 1988). Highland-fish endemism occurs throughout North America east of the Rocky Mountains (e.g., Table 1) but no comparative analysis has yet included the entire region. Here, we focus on six recently revised lineages that are distributed widely among North American highlands (Table 1) to (1) update and broaden hypotheses of relations among highlands and (2) present a hypothesis for cladogenesis.

## MATERIALS AND METHODS

We selected six clades of North American freshwater fishes (Euteleostei) for study based on presence of endemics among highlands throughout the study area and availability of recent, comprehensive phylogenetic hypotheses (Table 1) and followed Rosen's (1978) method of: (1) identifying endemic areas with endemic distributions and (2) discovering hierarchical, three-area relations to reveal sister areas (Parenti & Ebach, 2009). Disjunct highlands with endemic taxa were treated separately (Fig. 1). Sister taxa in the same highland area were grouped into a single, geographical taxon (Parenti & Ebach, 2009).
A three-area statement (e.g., if areas A, B, and C have the relationship A(BC), areas B and C are most closely related) is the smallest hierarchical statement in biogeography. Three-item analysis is the only cladistic-biogeographical method to use exclusive hierarchical hypotheses when constructing hypotheses of area relations (Cao *et al.*, 2007). It also filters missing and inapplicable data (Zaragüeta Bagils & Bourdon, 2007). In three-item analysis, three-area statements are combined to create optimal area cladograms, then an intersection tree is built from the three-area statements common to all optimal area cladograms (Cao *et al.*, 2009).

We used LisBeth (Laboratoire d'Informatique et Systématique program for Biogeography and Evolution using Three-item analysis; Zaragüeta Bagils et al., 2012) to conduct analyses. It applies fractional weighting (Nelson & Ladiges, 1992), paralogy-free subtree analysis (Nelson & Ladiges, 1996), and the transparent method (Ebach et al., 2005) to discover distributional congruence and resolve redundant areas (i.e., taxonomic paralogy) and widespread taxa (i.e., MASTs). The large number of areas analyzed (22) was accommodated by exporting three-area statements into a NEXUS matrix for analysis in Phylogenetic Analysis Using Parsimony software (PAUP, Swofford, 2003; Zaragüeta Bagils et al., 2012). PAUP was used to find optimal taxon-area cladograms, which were then imported into LisBeth to construct an intersection tree (Zaragüeta Bagils et al., 2012). Support for the validity of findings was estimated with two measures: (1) the retention index (Archie, 1989; Farris, 1989) is the percentage of three-area statements included in optimal area cladograms and (2) the completeness index (Zaragüeta Bagils et al., 2012) is the percentage of three-area statements present in optimal area cladograms that could be incorporated into the intersection tree. In each case, greater inclusion indicates greater congruence among taxa.

The intersection tree was interpreted using a comprehensive review of literature on geomorphic evolution of river drainages among highland areas. Only geological evidence exclusive of previous biogeographical findings was used so that the resulting hypothesis of highland-area relations would be independent from previous biogeographical hypotheses (Parenti & Ebach, 2009). A recent review of Gulf of Mexico drainage evolution by Galloway *et al.* (2011) was instrumental, but the literature was scoured for evidence relevant to the geomorphic evolution of river drainages and highland areas. A map representing pre-historic river drainages congruent with the intersection tree and compatible with previous estimates of lineage cladogenesis was constructed to provide an upgraded hypothesis of highland evolution and highland-fish cladogenesis.
## RESULTS

Ninety-one geographical taxa were included in this analysis (Appendix S1). Not all taxa of each clade are confined to highland areas, but endemism is primarily associated with highlands. Twenty-two highland areas that harbour endemic geographical taxa (Fig. 1a; Appendix S1) are distributed among major Atlantic and Gulf Coast river drainages from the Hudson River (USA) to the Ríos Nautla and Amería (México). The three-item analysis could not produce a final intersection tree until three widespread taxa were removed from the analysis. *Cyprinella venusta* "north and southwest" (as defined by Schönhuth & Mayden, 2010) occupies the Devils, Llano, Loess, Mobile, Pine, and Sabine highlands. *Cyprinella whipplei* "Mississippi tributaries" (as defined by Schönhuth & Mayden, 2010), occupies the Loess, Mobile, Moraine, Ohio, Ozark, and Tennessee highland areas. The other widespread taxon, a polytypic clade within *Oligocephalus* (Etheostoma) including *E. collettei*, *E. ditrema*, *E. nuchale*, and *E. swaini* (as presented in Near *et al.*, 2011), occupies the Apalachicola, Loess, Mobile, Ocala, Ouachita, and Pine highlands. Explanations for wide distributions and implications for this study are discussed below.

Three-item analysis identified 369 most-parsimonious trees with a retention index of 80.4%, which indicates substantial congruence within and among the fish clades analyzed. An intersection tree reconstructed from the three-area statements common among optimal taxon-area cladograms (Fig. 2) had a completeness index of 80.3%, demonstrating considerable congruence among most-parsimonious trees. Six major nodes identified seven sub-branches composed of one to eight highland areas each. Six sister-area relations were revealed. The Lerma highlands were excluded because only one geographical taxon was present (Appendix S1).

## Geological Interpretation and map explanation

The age of highland areas varies, but major highland uplifts (e.g., Appalachia, Ozark, Ouachita, Llano, Devils, Coahuila) were part of Palaeozoic Pangea (~330-260 ma) and remained tectonically active in the Mesozoic (~240-230 ma; Hatcher, 1989; Viele & Thomas, 1989; Harry & Londono, 2004). Thereafter, tectonism diminished except in the Basin and Range (Galloway *et al.*, 2011). River-drainage geography was dynamic because of subtle, in-situ tectonic events, active tectonism to the west, and climate change (Galloway *et al.*, 2011).
Fossil and molecular evidence indicate progenitors of at least some clades in this study arose in the Eocene-Oligocene period (Near et al., 2005, 2011). The Oligocene was a period of >10 ma with geomorphic stasis (relatively speaking) during which river-drainages established in the Late Eocene persisted (Galloway et al., 2011). Drainage divides were presumably barriers to fish dispersal and could have promoted initial cladogenesis. Thereafter, tectonic and climatic change in the Early and Middle Miocene caused major drainage rearrangement (Galloway et al., 2011). Emerging river drainages fragmented long-standing Oligocene drainages to create a new configuration that persisted for ~12 my (Galloway et al., 2000, 2011). Drainage rearrangement must have facilitated passive (e.g., stream capture) and active (e.g., corridor) geographic dispersal of ancestral highland fishes. Subsequent stability created new potential for allopatric speciation.

The basal node in the intersection tree (Fig. 2, node 1) corresponds to isolation of the Mobile highlands by formation of the Tennessee and Apalachicola rivers. The present course of the Tennessee River upstream of the Coastal Plain existed by Cretaceous time (Adams, 1928; Milici, 1968; Mills & Kaye, 2001) when erosional unroofing of the Appalachians began (Boettcher & Milliken, 1994). The Eocene Tennessee River discharged to the inundated Mississippi Embayment (Grim, 1936; Milici, 1968; Self, 2000). Middle Miocene uplift of the Blue Ridge and Cumberland Plateau, a cooler, more seasonal climate, and sea-level fall accelerated sedimentation (Boettcher & Milliken, 1994) and entrenched the upper Tennessee River. Retreating sea level and delta formation allowed the Tennessee River to extend southward onto the emerging Coastal Plain (Brown, 1967; Self, 2000, 2002). It reached the Continental Shelf in the Middle Miocene and continued in that course into the Pleistocene (Galloway et al., 2000, 2011; Fig. 1b). The modern Apalachicola River formed in the Middle Miocene when the Chattahoochee River (the west branch of the Apalachicola) beheaded the Tallapoosa River (Staheli, 1976; Galloway, 2008; Fig 1b). The area between the Tennessee and Apalachicola rivers, including the Black Warrior, Cahaba, Coosa, and Tallapoosa rivers of the Mobile highlands, was thus isolated.

The Black Warrior, Cahaba, Coosa, and Tallapoosa rivers maintained individual courses across the Coastal Plain (Fig. 1b) and deposited a continuous band of sediments along the coastline during the Late Miocene and Pliocene (Isphording, 1983; Otvos, 1998, 2004), but coalesced in the late Pliocene to form the Alabama River (Adams, 1929). The timing of coalescence corresponds to cessation of deposition along the coastline due to uplift, sea-level drop, and
climatic cooling (Otvos, 1998, 2004). Entrenchment of the Alabama River formed a drainage divide within Mio-Pliocene alluvial deposits (Maxwell, 1971) that separated the Mobile-Pine sister areas (Fig 2, sister relation 1).

Continued coastal uplift and increased, seasonal precipitation formed new coastal drainages (e.g., Escambia, Choctawhatchee) that dissected these deposits and created the Pine highlands (Price & Whetstone, 1977; Otvos, 1981, 1998). Sustained uplift precluded development of a coastal lowland (Price & Whetstone, 1977; Otvos, 2009), so river-drainage outlets remained proximate. By this time, the coastal band of Mio-Pliocene sediments extended west, to the Mississippi Embayment (Self 1986, 1993; Meylan & Li, 1995) and low sea-stands connected adjacent river drainages (Locker & Doyle, 1992), which presumably facilitated east-west fish dispersal. The Tennessee River was eventually captured by the Ohio River (Fig. 1a) and some remnants of the ancestral Lower Tennessee River joined the Mobile Bay drainage (Mobile highlands), whereas others were incorporated into the Pine highlands.

Emergence of Appalachian drainage divides accounts for two other main branches on the intersection tree (Fig. 2, nodes 3 and 4). Geographical relief was low across the Appalachians until uplift of the Blue Ridge and Cumberland Plateau, climatic cooling, and lowered sea levels of the Middle Miocene combined to amplify geographical relief (Cleaves, 1989; Stanford, 2010; Gallen et al., 2013). In response, developing river routes were rapidly entrenched (Bartholomew & Mills, 1991; Cleaves, 1993; Stanford et al., 2002). The energetic advantage of Atlantic Coast streams (i.e., shorter distance to sea level) allowed them to extend onto the Allegheny Plateau (Thompson, 1939, 1949). The erosion-resistant Blue Ridge became a major drainage divide in the Southern Appalachians (Hack, 1982).

Nevertheless, Atlantic Coast rivers still beheaded some interior streams (Hack, 1982; Prince et al., 2010, 2011) and the Savannah River captured the Chatooga River portion of the Chattahoochee-Apalachicola River (Staheli, 1976).

Thereafter, Atlantic Coast rivers draining to the Salisbury and Southeast Georgia embayments (Chesapeake and Georgia highlands, respectively) were isolated from Gulf of Mexico and Gulf of St. Lawrence drainages. Tributaries to the St. Lawrence River (e.g., Kentucky, New-Kanawha, Monongahela, Allegheny) were isolated northeast of the Cumberland Plateau, Blue Ridge, and Allegheny Plateau and flowed through the Appalachian Basin (Gray, 1991; Jacobson et al., 1988; Melhorn & Kempton, 1991; Kaktins & Delano, 1999; Fig. 1b).

As with the Mobile-Pine sister areas, the Chesapeake-Georgia and Apalachicola-Ocala sister areas (Fig. 2, sister relations 2 and 3) separated relatively recently. Uplift of the Cape Fear Arch in the Pliocene (Hack, 1982; Markewich,
1985) elevated the divide between the Chesapeake and Georgia highlands. To the south, land subsidence via dissolution began, in the Late Miocene, to uplift the Ocala Highlands (Opdyke et al., 1984; Adams et al., 2010).

Subsequent formation, by dissolution, of the Dougherty Plain and Solution Escarpment (Herrick & LeGrande, 1964) created an Apalachicola-Ocala divide (Fig. 1b). Mio-Pliocene incision of the Aucilla, Ochlockonee, and Suwanee rivers created the Ocala highlands (Otvos, 1981; Torak et al., 2010).

Association of the geographically disjunct Ohio and Apalachicola-Ocala highlands (Fig. 2) suggests similar ancestral traits were preserved in disjunct refugia. Glaciation explains the lack of a proximate sister area for the Ohio highlands.

Most of the St. Lawrence River drainage was repeatedly glaciated during the late Pliocene and Pleistocene, which isolated the unglaciated Ohio highlands. Adjacent areas that developed during interglacial periods and may have been colonized by Ohio-highland fishes (as they are today), but were subsequently re-glaciated.

Notably, the Ohio(Apalachicola-Ocala) relation (Fig. 2) reinforces historically debated evidence that Ohio-highland rivers were part of the pre-glacial St. Lawrence River drainage (Fig. 1b). The Kentucky and New-Kanawha rivers arose in the Middle Miocene and incision during regional uplift and climate change superimposed their courses across underlying strata (Bartholomew & Mills, 1991; Teller & Goldthwait, 1991). The Cincinnati Arch, which divides the Illinois and Appalachian basins (Sheets & Simonson, 2006), was the western boundary, except the Kentucky River was superimposed across it (Coffey, 1958; Melhorn & Kempton, 1991). Repeated glaciation formed proglacial lakes (Frolking et al., 2006; Fleeger et al., 2011) and diverted unglaciated headwaters into the Plio-Pleistocene Teays River (Gray, 1991; Melhorn & Kempton, 1991). Later (0.78 to 1.50 ma), the Ohio River breached the Madison Divide and rapidly incised its modern course (Pease & Gomez, 1997; Anthony & Granger, 2007).

The isolated position of the Ozark highlands (Fig. 2, node 2) corresponds to Late Tertiary isolation of the Upper Mississippi River drainage. Three pre-glacial rivers converged at Cap au Grès (Horberg, 1946; Rubey, 1952; Fig. 1b):
(1) the Platte-Des Moines River arose in the Northern Rocky Mountains and crossed the Central Great Plains (Witzke & Ludvigson, 1990; Wayne et al., 1991),
(2) the Mississippi River arose in the Superior Uplands and crossed the Illinois Basin (Anderson, 1988), with the Mahomet River as a major tributary (Kempton et al., 1991; Melhorn & Kempton, 1991),
and
(3) the Osage, Meramec, and Gasconade rivers arose in the High Plains and Boston Mountains and crossed the Ozark Plateaus (Aber, 1985, 1997; Elfrink & Siemens, 1998). Late Tertiary subsidence in the Northern Mississippi
Embayment (Ervin & McGinnis, 1975; Cox & Van Arsdale, 2002) and uplift of the Boston Mountains (McKeown et al., 1988) promoted erosion of the Ozark Plateaus and Illinois Basin (Gharrabi & Velde, 1995; Elfrink & Siemens, 1998) with increased deposition in the northwestern Mississippi Embayment (Potter, 1955; Van Arsdale et al., 2007). Ample sediment supply, aridity, and stable sea levels caused aggradation of a high alluvial surface that spanned much of the embayment (Guccione et al., 1990; Van Arsdale et al., 2007) and reduced output to the Gulf of Mexico (Galloway et al., 2011).

Aggradation in the northern Mississippi Embayment isolated the Ozark highlands (Fig. 1b). The only Appalachian river connected to the northern Mississippi Embayment was the Cumberland River (Anthony & Granger, 2007), which contributed to aggradation in the Northern Mississippi Embayment (Potter, 1955), but maintained a separate, easterly course (Fig. 1b) for hundreds of kilometres downstream (Guccione et al., 1990; Saucier, 1994; Van Arsdale et al., 2007). Connection to the Red River drainage (i.e., Ouachita highlands) was even more distant (Mange & Otvos, 2005; Galloway et al., 2011; Fig. 1b). The lack of an Ozark highland sister area is explained by Plio-Pleistocene glaciation, which subjected the Upper Mississippi River basin to glacial ice lobes (Anderson, 1988; Aber, 1991), periglacial permafrost (Péwé, 1983; Wayne et al., 1991), inundation by proglacial lakes (Todd, 1917), and meltwater floods (Aber, 1999; Kehew et al., 2009).

The Ouachita(Sabine-Loess) branch (Fig. 2, node 5) reflects Early Miocene isolation of the Ouachita highlands within the Red River drainage and proximity to the southern Mississippi Embayment and adjacent Gulf Coastal Plain (Galloway et al., 2011; Fig. 1b). Sedimentary deposits of the Mississippi, Red, and Sabine-Neches rivers accumulated along the Gulf Coast and in the southern Mississippi Embayment throughout the late Tertiary (Isphording & Flowers, 1983; Saucier, 1994). River courses frequently changed and merged (Saucier, 1994; Blum et al., 2000), creating passive and active dispersal opportunities. Dissection of Tertiary deposits commenced in response to regional uplift and Plio-Pleistocene climate change (Self, 1986; Saucier, 1994; Otvos, 2005). The Sabine-Loess sister areas (Fig 2, sister relation IV) likely separated during Pleistocene establishment of the Red-Mississippi river valley (Galloway et al., 2011), which presumably created a dispersal barrier.

The Tennessee-Moraine branch of the intersection tree (Fig. 2, node 6a, sister relation V) corresponds to Mio-Pliocene isolation of the Cumberland and Tennessee rivers (Potter, 1955; Brown, 1967; Galloway et al., 2011; Fig. 1b). Delta
deposits indicate the Tennessee River remained independent into the Pleistocene (Galloway *et al.*, 2011). The nascent Ohio River joined the Cumberland River ~1.5 ma (Granger *et al.*, 2001; Anthony & Granger, 2007; Galloway *et al.*, 2011) and presumably captured the Tennessee River thereafter, which provided a direct connection, via the Wabash and Upper Mississippi rivers, to the Moraine highlands. Later, the Mississippi River was diverted east of Crowley's Ridge and, eventually, into Thebes Gap (Blum *et al.*, 2000), where it even more closely connected to the Ohio-Cumberland-Tennessee River (Fig. 1a).

The remaining eight branches composed the crown of the intersection tree, which includes all southwestern highland areas (Fig. 2, node 6b). Middle Miocene dismemberment of ancient Brazos and Rio Grande drainages created potential east-west stream connections from the Mississippi Embayment to the Tampico-Misantla Basin, but tectonism and climate change thereafter caused river-drainage fragmentation and long-term isolation (Galloway *et al.*, 2011; Fig. 1b). Uplift of the Llano and Capitan highlands (Hill, 1996; Cather *et al.*, 2012) created the Miocene Guadalupe River (Galloway *et al.*, 2011) and isolated the peripheral Cabeza de Vaca, Parras, and Tampico highlands.

These disjunct areas form an unresolved (i.e., polytomic) branch of the crown group. Remaining highlands became isolated in the Late Miocene and Pliocene as tectonism and climate change fragmented the Río Bravo, Rio Grande, and Guadalupe drainages (Galloway *et al.*, 2011). Early Miocene faulting and aridity isolated the Conchos highlands in the Chihuahua Trough (King & Adkins, 1946; Haenggi, 2002; Galloway *et al.*, 2011). The Capitan and Llano highlands (a polytomic pair) were part of the Guadalupe River until Late Miocene dissolution-subsidence diverted streams of the Capitan highlands into the Delaware Basin (Maley & Huffington, 1953; Hawley, 1993a, 1993b). Isolation of the Coahuila-Devils sister highlands occurred simultaneously via faulting along the Balcones Escarpment (Kastning, 1983; Barker *et al.*, 1994). Finally, faulting in the Coahuila Folded Belt and uplift of the Tamaulipas Arch (Aranda-Gómez *et al.*, 2005; Chávez-Cabello *et al.*, 2007) separated the Coahuila-Devils sister areas (Fig. 2, sister relation VI).

Highland isolation peaked in the Late Miocene and early Pliocene (Galloway *et al.*, 2011; Fig. 1b). Subsequent climatic cooling re-connected drainage fragments to create "mega-drainages" (sensu Blum & Hattier-Womack, 2009). The Mega Mississippi River, a major conduit for glacial runoff, became the dominant Gulf of Mexico drainage (Saucier, 1994; Blum *et al.*, 2000) and connected the Ozark, Ohio, Loess, Ouachita, and Tennessee and (interglacial) Moraine highlands. The Mega Mobile Bay drainage integrated the Black Warrior, Cahaba, Coosa, and Tallapoosa rivers
(Adams, 1929). The Mega Rio Grande initially connected the Parras, Conchos, Devils, Capitan, Coahuila highlands (Strain, 1970; Hawley, 1993a; Hill, 1996; Mack et al., 2006), but tectonism and aridity later disconnected the Parras highlands (Albritton, 1958). The Mega Brazos River integrated the Calcasieu, Sabine, Trinity, and Brazos rivers, but high sea levels in interglacial periods (such as the present) caused re-fragmentation (Blum & Hattier-Womack, 2009).

# DISCUSSION

## Methodology

Use of three-item analysis increases potential to discover new patterns (Parenti & Ebach, 2009, 2013) and, in this case, to update existing biogeographical hypotheses without relying on existing interpretations. Incorporation of hierarchical, phylogenetic hypotheses embeds evolutionary hypotheses into the analysis, such that three-item analysis reveals area relations based on evolutionary relations, not just shared traits (Williams & Ebach, 2006; Cao et al., 2007). This allows more direct interplay (i.e., reciprocal illumination) between biological and geological evidence. Nevertheless, this study supports many existing biogeographical hypotheses. For instance, the Mobile highlands were basal to all others in this study and in previous ones (Mayden, 1988; Crandall & Templeton, 1999). However, inclusion of many more highland areas in this study revealed that ancient highlands are often more closely related to younger highlands of the Coastal Plain than to other ancient highlands. In particular, the Ouachita and Ozark highlands were distantly related, consistent with long-standing, pre-glacial isolation.

Subdivision of some areas used in this study would be beneficial because area relations are most informative when three-area relationships are revealed (Parenti & Ebach, 2009). For example, the Ohio and Ozark highlands each lacked sister areas, so separate analysis of drainages of the Ohio highlands (Green, Kanawha, Kentucky, Monongahela) and Ozark highlands (Gasconade, Meramec, Neosho, Osage, White) would more accurately reveal area relations.

Further, evidence revealed during map construction suggests the Cumberland, Tennessee, Black Warrior, Cahaba, Coosa, and Tallapoosa rivers should be analyzed separately because each was independent during the Late Tertiary.

Biological evidence supports long separation of these drainages (e.g., Starnes & Etnier, 1986; Mayden, 1987; Near & Keck, 2005; Campbell et al., 2008). Given this, it would also be useful to divide the Pine highlands into separate east (ancestral Black Warrior, Cahaba, Coosa, and Tallapoosa rivers) and west (Tennessee River) sections.
A possible weakness of three-item analysis is that it provides only one hypothesis for highland-area relations when more than one pattern may be present. For example, this study identified a sister relation between the Tennessee and Moraine highlands, reflecting colonization of deglaciated regions by fishes related closely to Tennessee highland endemics. However, it is well established that fishes re-colonized glaciated regions from several refugia (e.g., Hoagstrom & Berry, 2006; Berendzen *et al.*, 2008). Thus, a comparative analysis using different clades could discover a different sister area to the Moraine highlands. A potential solution will be to conduct additional studies with different combinations of taxa until a suite of representative patterns is identified.

The goal of three-item analysis is to discover shared patterns (Parenti & Ebach, 2009, 2013), which precludes the use of a priori manipulations, such as time slicing (sensu Upchurch & Hunn, 2002). Hence, patterns reported here could include some reticulate area relations. This does not invalidate these relations and, in fact, multiple cycles of speciation that demonstrate similar area relations are strong evidence for a significant biogeographical pattern. In an effort to devise an accurate geobiotic scenario it is ultimately necessary to distinguish a historical sequence of biogeographical events (Morrone, 2009). The broad geographic scale of this study allowed many independent and diverse geological studies to be incorporated for interpretation of the intersection tree. The fact that major nodes correspond to geographically disjunct Middle Miocene to Pliocene events is strong evidence that this period was significant for highland-fish cladogenesis. As more rigorously dated phylogenies (e.g., Near *et al.*, 2003, 2005) become available, it will be possible to better correlate speciation events with rigorously dated geological events (sensu Hovenkamp, 2001; Fattorini, 2008).
Synthesis - Ecological Biogeography of highland-fish cladogenesis

It is important to connect biogeographical patterns with ecological paradigms to provide a context for hypothesis generation (Brown *et al.*, 1996). Patterns of cladogenesis in highland fishes resemble a taxon cycle in which species proceed through four stages: (1) geographic expansion, (2) population contraction, isolation, and differentiation, (3) range fragmentation and population extirpation, and (4) restriction to a single area (Ricklefs & Cox, 1972; Ricklefs & Bermingham, 1999). Stage 1 species are colonists from neighbouring regions or derived residents that can expand their distribution through biological release from predators or pathogens (i.e., a conquistador effect). Species in later stages no longer sustain biological release, are more ecologically specialized, and are increasingly confined to interior, high-elevation habitats, in part by low-elevation biological interactions (Brown *et al.*, 1996). Late-stage species persist longer in larger, more insular habitats where they are less vulnerable to stage-1 species and stochastic extirpation (MacArthur & Wilson, 1967; Cox & Ricklefs, 1972).

Evidence consistent with taxon-cycle predictions is present throughout highland-fish literature. For example,
ancestral *Campostoma spadiceum* and *Lythrurus snelsoni* of the Ouachita highlands (Pramuk *et al.*, 2007; Blum *et al.*,
2008; Cashner *et al.*, 2010) are putative stage 4 species that originated in the late Tertiary when the Ouachita
highlands were unroofed and Ouachita streams were isolated. These species are confined to highland-type habitats
and have parapatric distributions with younger, widespread (putative stage 1) *Campostoma plumbeum* and *Lythrurus
umbratilis*, respectively (Taylor & Lienesch, 1996; Cashner *et al.* 2010). *Lythrurus umbratilis* has even colonized
highland-type habitats lacking *L. snelsoni* (Taylor & Lienesch, 1996). *Notropis suttkusi*, another putative stage 4
Ouachita-highland endemic, is also restricted to highland habitats, although no close relatives have colonized the area
(Berendzen *et al.*, 2008).

In general, younger (Quaternary) highland taxa are more widespread than pre-glacial (Tertiary) taxa (Pflieger, 1971; Near & Benard, 2004; Roe et al., 2008). Each clade in this study included widespread, putative stage 1 taxa that are not restricted to rocky, highland-type habitats (Table 2). Some, such as *Cyprinella lutrensis* (Walters et al., 2008; Gido & Propst, 2012) and *Micropterus dolomieu* (Hoagstrom et al., 2007b; Stefferud et al., 2011), are aggressive non-native invaders, which supports the hypothesis of stage-1 biological release (Ricklefs & Bermingham, 1999). Geographical differentiation in some widespread taxa (e.g., Lang & Echelle, 2011; Husemann et al., 2012) suggests they have
progressed to stage 2. Widespread, geographic taxa excluded from this analysis (see results) are likely in stage 1 or stage 2, in which case their distributions should be incongruent with stage 3 and 4 highland endemics. This could explain why their distributions were incongruent with the dominant distributional pattern.

Geological evidence suggests stage 1 progenitors of Tertiary highland endemics could have ranged widely across aggraded, North American landscapes from the Late Eocene to the Early Miocene, consistent with Pflieger (1971) and Mayden (1987, 1988) who hypothesized that an extensive, pre-glacial upland allowed expansion of highland-fish populations. River-channel avulsion and cross grading are common in aggrading areas (e.g., Phillips, 2009) and create inter-drainage connections and stream transfers that must help fishes disperse. In particular, large-scale inter-drainage connections during Middle Miocene drainage rearrangements would have promoted broad geographic spread. Thereafter, uplift and accelerated erosion created novel, sediment-starved, highland habitats that are ecologically unique (e.g., Jenkins & Freeman, 1972; Wilkinson & Edds, 2001; Walters et al., 2003). Highland endemics typically require high stream gradients, low turbidity, stable substrata, and stable water chemistry (e.g., Braasch & Smith, 1965; Taylor & Lienesch, 1995, 1996; Brewer et al., 2007). Molecular evidence supports Late Miocene speciation in at least some highland-fish clades (Near et al., 2003, 2011; Near & Benard, 2004; Webb et al., 2004; Near and Keck, 2005; Domínguez-Domínguez et al., 2011). Descendants of some lowland-type fishes also speciated in highlands at the same time (e.g., Echelle et al., 2005; Niemiller et al., 2012). Small body size (Hardman & Hardman, 2008), discrete sexual selection aided by high water clarity (Mendelson et al., 2007), and character displacement by sympatric congeners (Carlson et al., 2009) potentially accelerated speciation.

Late Miocene to Pliocene resurgence of aggradation would have isolated highland specialists and prolonged isolation would have produced stage-3 species. Increasing aridity could have caused extirpation of some stage 3 populations, especially in smaller highland areas (sensu Hoagstrom & Berry, 2006), with remnant taxa restricted to a single area representing stage-4 species. The boundaries of highland-fish distributions often correspond to geomorphic knick points (i.e., falls and rapids) that form a transition between lowland and upland habitats (Hack, 1982). These ecological and, sometimes, physical barriers not only form the downstream boundary of highland-type habitats, but also can cause dispersal limitation that maintains depauperate faunas (Hubbell, 2001; Hoagstrom et al., 2007a). But, knick points are dynamic erosional features that advance inland over time as highlands are converted to lowlands
(Frankel et al., 2007). Thus, highland refuges can shrink over time, which increases susceptibility to stochastic events (MacArthur & Wilson, 1967; Ricklefs and Cox, 1972).

For example, the New River Plateau is a shrinking highland refuge. Some headwaters of the New River have been captured by the Roanoke River (Prince et al., 2011) and downstream knick points are eroding headward at a geologically rapid rate (Prince & Spotila, 2013). Biogeographical evidence suggests other headwaters of the New River drainage were captured by Cumberland or Tennessee river tributaries, although a New River tributary did capture a tributary to the Monongahela River (Jenkins, 1994). In each case, the drainage with a more direct route to sea level had the advantage and, if these processes continue apace, endemics of the New River Plateau will have to survive transfer to other river drainages or invasion by non-endemics to persist. This may be an extreme example, but similar processes occur throughout the study area.

But, emerging alluvial uplands, retreating knick points, and headwater captures make vast, growing territories for stage-1 species and subsequent erosion and dissection of aggraded landscapes produces new highland areas. For example, geological evidence indicates the taxon cycle in Coastal Plain highlands must be relatively recent in response to Plio-Pleistocene evolution of highland-type landscapes (Otvos, 2009; Stults et al., 2010). Late Pleistocene divergence between *Percina kathae* of the Mobile highlands and *P. austroperca* of the Pine highlands (Near & Benard, 2004) is congruent with dispersal of a stage-1 progenitor in the Late Miocene and early Pliocene followed by Plio-Pleistocene separation of these sister areas and putative sister species. Multiple sister-relations of continental highlands with adjacent Coastal Plain highlands in this study indicate geographic dispersal of highland fishes was more restricted in the Plio-Pleistocene than in the Early Miocene, consistent with predictions of the taxon cycle (Ricklefs & Cox, 1978; Ricklefs & Bermingham, 1999).

General cladogenesis among highland areas indicates parallel evolution or (more specifically) parallel taxon cycles. In the same way that anadromous fishes diversify as they colonize post-glacial landscapes (Bell & Andrews, 2007), widespread highland-type fishes apparently diversify as they colonize highland habitats. Plethodontid salamanders exhibit similar patterns (Kozak et al., 2009). This model of speciation has been described as a "raceme" (Bell & Foster, 1994) or "flexible stem" (Wund et al., 2008) wherein a widespread progenitor with high standing genetic diversity or high developmental plasticity gives rise to similar divergent lineages among disjunct, ecological similar areas.
However, highlands are much older than post-glacial landscapes, so highland fishes could diverge far from the progenitor or even outlive it. Like highland salamanders (Kozak & Wiens, 2006), endemic highland fishes exhibit niche conservatism, but emerging (stage 1) species continue to colonize highlands as dispersal opportunities allow, which allows endemic faunas of mixed age to accumulate over time (sensu Hoagstrom & Berry, 2006).
## ACKNOWLEDGEMENTS

We thank Michael J. Blum, David A. Etnier, Phillip R. Hollingsworth, Jr., and C. Darrin Hulsey for information on fish distributions and Andrew K. Rindsberg for helpful discussion on ancestral Tennessee and Mobile Bay rivers.

## REFERENCES

Aber, J.S. (1985) Quartzite-bearing gravels and drainage development in eastern Kansas. *TER-QUA Symposium Series*, **1**, 105-110.

Aber, J.S. (1991) The glaciation of northeastern Kansas. *Boreas*, **20**, 297-314.

Aber, J.S. (1997) Chert gravel and neogene drainage in east-central Kansas. *Kansas Geological Survey Bulletin*, **240**, 29-41.

Aber, J.S. (1999) Pre-Illinoian glacial geomorphology and dynamics in the central United States, west of the Mississippi. *Geological Society of America Special Paper*, **337**, 113-119.

Adams, G.I. (1928) The course of the Tennessee River and the physiography of the southern Appalachian region. *Journal of Geology*, **36**, 481-493.

Adams, G.I. (1929) The streams of the Coastal Plain of Alabama and the Lafayette problem. *Journal of Geology*, **37**, 193-203.

Adams, P.N., Opdyke, N.D. & Jaeger, J.M. (2010) Isostatic uplift drive by karstification and sea-level oscillation: modelling landscape evolution in north Florida. *Geology*, **38**, 531-534.

Albritton, C.C.Jr. (1958) Quaternary stratigraphy of the Guadiana Valley, Durango, Mexico. *Geological Society of America Bulletin*, **69**, 1197-1216.

Anderson, R.C. (1988) Reconstruction of preglacial drainage and its diversion by earliest glacial forebulge in the upper Mississippi Valley region. *Geology*, **16**, 254-257.

Anthony, D.M. & Granger, D.E. (2007) A new chronology for the age of Appalachian erosional surfaces determined by cosmogenic nuclides in cave sediments. *Earth Surface Processes and Landforms*, **32**, 874-887.
Aranda-Gómez, J.J., Housh, T.B., Luhr, J.F., Henry, C.D., Becker, T. & Chávez-Cabello, G. (2005) Reactivation of the San Marcos fault during mid-to-late Tertiary extension, Chihuahua, Mexico. *Geological Society of America Special Paper*, **393**, 509-521.

Archie, J.W. (1989) Homoplasy excess ratios: new indices for measuring levels of homoplasy in phylogenetic systematics and a critique of the consistency index. *Systematic Zoology*, **38**, 253-269.

Bagley, J.C., Mayden, R.L., Roe, K.J., Holznagel, W. & Harris P.M. (2011) Congeneric phylogeographical sampling reveals polyphyly and novel biodiversity within black basses (Centrarchidae: *Micropterus*). *Biological Journal of the Linnean Society*, **104**, 346-363.

Barker, R.A., Bush, P.W. & Baker, E.T.Jr. (1994) Geologic history and hydrogeologic setting of the Edwards-Trinity aquifer system, west-central Texas. *U.S. Geological Survey Water-Resources Investigations Report*, **94-4039**, 1-51.

Bartholomew, M.J. & Mills, H.H. (1991) Old courses of the New River: its late Cenozoic migration and bedrock control inferred from high-level stream gravels, southwestern Virginia. *Geological Society of America Bulletin*, **103**, 73-81.

Bell, M.A. & Andrews C.A. (1997) Evolutionary consequences of postglacial colonization of fresh water by primitively anadromous fishes. *Evolutionary ecology of freshwater animals* (ed. by B. Streit, T Städler, & C.M. Lively), pp. 323-363.

Birkhäuser Verlag, Basel, Switzerland.

Bell, M.A. & Foster, S.A. (1994) Introduction to the evolutionary biology of the threespine stickleback. *The evolutionary biology of the threespine stickleback* (ed. by M.A. Bell & S.A. Foster), pp. 1-27. Oxford University Press, New York.

Berendzen, P.B., Simons, A.M., Wood, R.M., Dowling, T.E. & Secor, C.L. (2008) Recovering cryptic diversity and ancient drainage patterns in eastern North America: historical biogeography of the *Notropis rubellus* species group (Teleostei: Cypriniformes). *Molecular Phylogenetics and Evolution*, **46**, 721-737.

Blum, M.D. & Hattier-Womack, J. (2009) Climate change, sea-level change, and fluvial sediment supply to deepwater depositional systems. *SEPM Special Publication*, **92**, 15-39.

Blum, M.D., Guccione, M.J., Wysocki, D.A., Robnett, P.C. & Rutledge, E.M. (2000) Late Pleistocene evolution of the lower Mississippi River valley, southern Missouri to Arkansas. *Geological Society of America Bulletin*, **112**, 221-235.
Blum, M.J., Neely, D.A., Harris, P.M. & R.L. Mayden (2008) Molecular systematics of the cyprinid genus *Campostoma* (Actinopterygii: Cypriniformes): disassociation between morphological and mitochondrial differentiation. *Copeia*, **2008**, 360-369.

Boettcher, S.S. & Milliken, K.L. (1994) Mesozoic-Cenozoic unroofing of the southern Appalachian Basin: apatite fission track evidence from middle Pennsylvanian sandstones. *Journal of Geology*, **102**, 655-668.

Braasch, M.E. & Smith, P.W. (1965) Relationships of the topminnows *Fundulus notatus* and *Fundulus olivaceus* in the upper Mississippi River valley. *Copeia*, **1965**, 46-53.

Brewer, S.K., Rabeni, C.F., Sowa, S.P. & Annis G. (2007) Natural landscape and stream segment attributes influencing the distribution and relative abundance of riverine smallmouth bass in Missouri. *North American Journal of Fisheries Management*, **27**, 326-341.

Brown, B.W. (1967) A Pliocene Tennessee River hypothesis for Mississippi. *Southeastern Geology*, **8**, 81-84.

Brown, J.H., Stevens, G.C. & Kaufman, D.M. (1996) The geographic range: size, shape, boundaries, and internal structure. *Annual Review of Ecology and Systematics*, **27**, 597-623.

Cao, N., Zaragüeta Bagils, R. & Vignes-Lebbe, R. (2007) Hierarchical representation of hypotheses of homology.
*Geodiversitas*, **29**, 5-15.

Carlson, R.L., Wainwright, P.C. & Near, T.J. (2009) Relationship between species co-occurrence and rate of morphological change in *Percina* darters (Percidae: Etheostomatinae). *Evolution*, **63**, 767-778.

Campbell, D.C., Johnson, P.D., Williams, J.D., Rindsberg, A.K., Serb, J.M., Small, K.K. & Lydeard, C. (2008) Identification of 'extinct' freshwater mussel species using DNA barcoding. *Molecular Ecology Resources*, **8**, 711-724.

Cashner, R.C., Matthews, W.J., Marsh-Matthews, E., Unmack, P.J. & Cashner, F.M. (2010) Recognition and redescription of a distinctive stoneroller form the southern interior highlands. *Copeia*, **2010**, 300-311.

Cather, S.M., Chapin, C.E., & Kelley, S.A. (2012) Diachronous episodes of Cenozoic erosion in southwestern North America and their relationship to surface uplift, paleoclimate, paleodrainage, and paleoaltimetry. *Geosphere*, **8**, 1177-1206.
Chávez-Cabello, G., Aranda-Gómez, J.J., Molina-Garza, R.S., Cossío-Torres, T., Arvizu-Gutiérrez, I.R. & González-Naranjo, G.A. (2007) The San Marco fault: a Jurassic multireactivated basement structure in northeastern México. Geological Society of America Special Paper, 422, 261-286.

Cleaves, E.T. (1989) Appalachian Piedmont landscapes from the Permian to the Holocene. *Geomorphology*, **2**, 159-179.

Cleaves, E.T. (1993) Climatic impact on isovolumetric weathering of a coarse-grained schist in the northern Piedmont Province of the central Atlantic states. *Geomorphology*, **8**, 191-198.

Coffey, G.N. (1958) Major glacial drainage changes in Ohio. *Ohio Journal of Science*, **58**, 43-49.

Cox, R.T. & Van Arsdale, R.B. (2002) The Mississippi Embayment, North America: a first order continental structure generated by the Cretaceous superplume mantle event. *Journal of Geodynamics*, **34**, 163-176.

Crandall, K.A. & Templeton A.R. (1999) The zoogeography and centers of origin of the crayfish subgenus Procericambarus (Decapoda: Cambaridae). *Evolution*, **53**, 123-134.

Domínguez-Domínguez, O., Vila, M., Pérez-Rodríguez, R., Remón, N. & Doadrio, I. (2011) Complex evolutionary history of the Mexican stoneroller *Campostoma ornatum* Girard, 1856 (Actinopterygii: Cyprinidae). *BMC Evolutionary Biology*, **11**, 153.

Doosey, M.H., Bart, H.L.Jr., Saitoh, K. & Miya, M. (2010) Phylogenetic relationships of catostomid fishes (Actinopterygii: Cypriniformes) based on mitochondrial ND4/ND5 gene sequences. *Molecular Phylogenetics and Evolution*, **54**, 1028-1034.

Ebach, M.C., Humphries, C.J., Newman, R.A., Williams, D.M. & Walsh, S.A. (2005) Assumption 2: opaque or intuition? *Journal of Biogeography*, **32**, 781-787.

Echelle, A.A., Carson, E.W., Echelle, A.F., Van Den Bussche, R.A., Dowling, T.A. & Meyer, A. (2005) Historical biogeography of the new-world pupfish genus *Cyprinodon* (Teleostei: Cyprinodontidae). *Copeia*, **2005**, 320-339.

Elfrink, N.M. & Siemens, M.A. (1998) Quaternary drainage shifts in Missouri. *Guidebook for the 45th Annual Field Trip*, pp. 48-69. Association of Missouri Geologists, Farmington, Missouri.

Ervin, C.P. & McGinnis, L.D. (1975) Reelfoot Rift: reactivated precursor to the Mississippi Embayment. *Geological Society of America Bulletin*, **86**, 1287-1295.
Farris, J.S. (1989) The retention index and the rescaled consistency index. *Cladistics*, **5**, 417-419.

Fattorini, S. (2008) Hovenkamp's ostracized vicariance analysis: testing new methods of historical biogeography.
*Cladistics*, **24**, 611-622.

Fleeger, G.M., Grote, T., Straffin, E. & Szabo, J.P. (2011) Quaternary geology of northwestern Pennsylvania. *Geological Society of America Field Guide*, **20**, 87-109.

Frankel, K.L., Pazzaglia, F.J. & Vaughn, J.D. (2007) Knickpoint evolution in a vertically bedded substrate, upstream-dipping terraces and Atlantic slope bedrock channels. *Geological Society of America Bulletin*, **119**, 476-486.

Frolking, T.A & Pachell, M.A. (2006) Glacial Lake Licking: late-glacial drainage diversion and the formation of Black Hand Gorge, Licking County, Ohio. *Ohio Journal of Science*, **106**, 103-111.

Gallen, S.F., Wegmann, K.W. & Bohnenstiehl, D.R. (2013) Miocene rejuvenation of topographic relief in the southern Appalachians. *GSA Today*, **23**, 4-10.

Galloway, W.E. (2008) Depositional evolution of the Gulf of Mexico sedimentary basin. *Sedimentary Basins of the World*, **5**, 505-549.

Galloway, W.E., Whiteaker, T.L. & Ganey-Curry, P. (2011) History of Cenozoic North American drainage basin evolution, sediment yield, and accumulation in the Gulf of Mexico basin. *Geosphere*, **7**, 938-973.

Galloway, W.E., Ganey-Curry, P.E., Li, X. & Buffler, R.T. (2000) Cenozoic depositional history of the Gulf of Mexico basin. *AAPG Bulletin*, **84**, 1743-1774.

Gharrabi, M. & Velde, B. (1995) Clay mineral evolution in the Illinois Basin and its causes. *Clay Minerals*, **30**, 353-364.

Gido, K.B. & Propst, D.L. (2012) Long-term dynamics of native and nonnative fishes in the San Juan River, New Mexico and Utah, under a partially managed flow regime. *Transactions of the American Fisheries Society*, **141**, 645-659.

Granger, D.E., Fabel, D. & Palmer, A.N. (2001) Pliocene-Pleistocene incision of the Green River, Kentucky, determined from radioactive decay of cosmogenic $^{26}$Al and $^{10}$Be in Mammoth Cave sediments. *Geological Society of America Bulletin*, **113**, 825-836.

Gray, H.H. (1991) Origin and history of the Teays drainage system; the view from midstream. *Geological Society of America Special Paper*, **259**, 43-50.

Grim, R.E. (1936) The Eocene sediments of Mississippi. *Mississippi Geological Survey Bulletin*, **30**, 1-240.
Guccione, M.J., Prior, W.L. & Rutledge, E.M. (1990) The Tertiary and early Quaternary geology of Crowley's Ridge.

*Field Guide to the Mississippi Alluvial Valley, Northeast Arkansas and Southeast Missouri* (ed. by MJ Guccione and EM Rutledge), pp. 23-44. Friends of the Pleistocene, South-Central Cell.

Hack, J.T. (1982) Physiographic divisions and differential uplift in the Piedmont and Blue Ridge. *U.S. Geological Survey Professional Paper, 1265*, 1-49.

Haenggi, W.T. (2002) Tectonic history of the Chihuahua trough, Mexico and adjacent USA, Part II: Mesozoic and Cenozoic. *Boletín de la Sociedad Geológica Mexicana*, **55**, 38-94.

Hardman, M. & Hardman L.M. (2008) The relative importance of body size and paleoclimatic change as explanatory variables influencing lineage diversification rate: an evolutionary analysis of bullhead catfishes (Siluriformes: Ictaluridae). *Systematic Biology*, **57**, 116-130.

Harry, D.L. & Londono, J. (2004) Structure and evolution of the central Gulf of Mexico continental margin and coastal plain, southeast United States. *Geological Society of America Bulletin*, **116**, 188-199.

Hatcher, R.D.Jr. (1989) Tectonic synthesis of the U.S. Appalachians. *The Appalachian-Ouachita orogen in the United States* (ed. by R.D. Hatcher, Jr., W.A. Thomas, & G.W. Viele), pp. 511-535. Geological Society of America, Boulder, Colorado.

Hawley, J.W. (1993a) Overview of the geomorphic history of the Carlsbad area. *New Mexico Geological Society, Fall Field Conference Guidebook*, **44**, 2-3.

Hawley, J.W. (1993b) The Ogallala and Gatuna formations in the southeastern New Mexico region, a progress report. *New Mexico Geological Society, Fall Field Conference Guidebook*, **44**, 261-269.

Herrick, S.M. & LeGrand, H.E. (1964) Solution subsidence of a limestone terrane in southwest Georgia. *International Association of Scientific Hydrology Bulletin*, **9**, 25-36.

Highton, R. (1995) Speciation in eastern North American salamanders of the genus *Plethodon*. *Annual Review of Ecology and Systematics*, **26**, 579-600.

Hill, C.A. (1996) Geology of the Delaware Basin, Guadalupe, Apache, and Glass Mountains, New Mexico and west Texas. *Permian Basin Section-SEPM Publication*, **96-39**, 1-480.
Hoagstrom, C.W. & Berry, C.R.Jr. (2006) Island biogeography of native fish faunas among Great Plains drainage basins: basin scale features influence composition. *American Fisheries Society Symposium*, **48**, 221-264.

Hoagstrom, C.W., Gosch, N.J.C., DeWitte, A.C., Berry, C.R.Jr., & Duehr, J.P. (2007a) Biodiversity, Biogeography, and longitudinal fish faunal structure among perennial, warmwater streams of the Cheyenne River drainage. *Prairie Naturalist*, **39**, 117-144.

Hoagstrom, C.W., DeWitte, A.C., Gosch, N.J.C. & Berry C.R. Jr. (2007b) Historical fish assemblage flux in the Cheyenne River below Angostura Dam. *Journal of Freshwater Ecology*, **22**, 219-229.

Horberg, L. (1946) Preglacial erosion surfaces in Illinois. *Journal of Geology*, **54**, 179-192.

Hovenkamp, P. (2001) A direct method for the analysis of vicariance patterns. *Cladistics*, **17**, 260-265.

Hubbell, S.P. (2001) *The unified neutral theory of biodiversity and biogeography*. Princeton University Press, Princeton.

Husemann, M., Ray, J.W., King, R.S., Hooser, E.A. & Danley, P.D. (2012) Comparative biogeography reveals differences in population genetic structure of five species of stream fishes. *Biological Journal of the Linnean Society*, **107**, 867-885.

Isphording, W.C. (1983) Interpretive mineralogy: examples from Miocene coastal plain sediments. *Gulf Coast Association of Geological Societies Transactions*, **33**, 295-305.

Isphording, W.C. & Flowers, G.C. (1983) Differentiation of unfossiliferous clastic sediments: solutions from the southern portion of the Alabama-Mississippi Coastal Plain. *Tulane Studies in Geology and Paleontology*, **17**, 59-83.

Jacobson, R.B., Elston, D.P. & Heaton, J.W. (1988) Stratigraphy and magnetic polarity of the high terrace remnants in the upper Ohio and Monongahela rivers in West Virginia, Pennsylvania, and Ohio. *Quaternary Research*, **29**, 216-232.

Jenkins, R.E. (1980) *Moxostoma duquesnii* (Lesueur), black redhorse. Atlas of North American Freshwater Fishes (ed. by D.S. Lee, C.R. Gilbert, C.H. Hocutt, R.E. Jenkins, D.E. McAllister & J.R. Stauffer, Jr.), pp. 419-420. North Carolina State University Museum of Natural History, Raleigh.

Jenkins, R.E. (1994) Biogeography. *Freshwater Fishes of Virginia* (R.E. Jenkins & N.M. Burkhead), pp. 43-99. American Fisheries Society, Bethesda, Maryland.

Jenkins, R.E. & Freeman, C.A. (1972) Longitudinal distribution and habitat of fishes of Mason Creek, and upper Roanoke River drainage tributary, Virginia. *Virginia Journal of Science*, **23**, 194-202.
Kaktins, U. & Delano, H.L. (1999) Drainage basins. *The geology of Pennsylvania* (ed. by A.P. Schultz), pp. 379-390.

Pennsylvania Geological Survey and Pittsburgh Geological Society, Harrisburg.

Kastning, E.H. (1983) Relict caves as evidence of landscape and aquifer evolution in a deeply dissected carbonate terrain: southwest Edwards Plateau, Texas, U.S.A. *Journal of Hydrology*, **61**, 89-112.

Kehew, A.E., Lord, M.L., Kozlowski, A.L. & Fisher T.G. (2009) Proglacial megaflooding along the margins of the Laurentide ice sheet. *Megaflooding on Earth and Mars* (ed. by D.M. Burr, P.A. Carling & V.R. Baker), pp. 104-127.

Kempton, J.P., Johnson, W.H., Heigold, P.C. & Cartwright, K. (1991) Mahomet Bedrock Valley in east-central Illinois; topography, glacial drift stratigraphy, and hydrogeology. *Geological Society of America Special Paper* **258**, 91-124.

King, R.E. & Adkins, W.S. (1946) Geology of a part of the lower Conchos Valley, Chihuahua, Mexico. *Geological Society of America Bulletin*, **57**, 275-294.

Kozak, K.H. & Wiens, J.J. (2006) Does niche conservatism promote speciation? A case study in North American salamanders. *Evolution*, **60**, 2604-2621.

Kozak, K.H., Mendyk, R.W. & Wiens, J.J. (2009) Can parallel diversification occur in sympatry? Repeated patterns of body-size evolution in coexisting clades of North American salamanders. *Evolution*, **63**, 1769-1784.

Lang, N.J. & Echelle, A.A. (2011) Novel phylogeographic patterns in a lowland fish, *Etheostoma proeliare* (Percidae). *Southeastern Naturalist*, **10**, 133-144.

Locker, S.D. & Doyle, L.J. (1992) Neogene to recent stratigraphy and depositional regimes of the northwest Florida inner continental shelf. *Marine Geology*, **104**, 123-138.

MacArthur, R.H. & Wilson, E.O. (1967) *The theory of island biogeography*. Princeton University Press, Princeton.

Mack, G.H., Seager, W.R., Leeder, M.R., Perez-Arlucea, M. & Salyards, S.L. (2006) Pliocene and Quaternary history of the Rio Grande, the axial river of the southern Rio Grande rift, New Mexico, USA. *Earth-Science Reviews*, **79**, 141-162.

Maley, V.C. & Huffington, R.M. (1953) Cenozoic fill and evaporate solution in the Delaware Basin, Texas and New Mexico. *Geological Society of America Bulletin*, **64**, 539-546.

Mange, M.A. & Otvos, E.G. (2005) Gulf coastal plain evolution in west Louisiana: heavy mineral provenance and Pleistocene alluvial chronology. *Sedimentary Geology*, **182**, 29-57.
Markewich, H.W. (1985) Geomorphic evidence for Pliocene-Pleistocene uplift in the area of the Cape Fear Arch, North Carolina. *Tectonic Geomorphology* (ed. by M. Morisawa & J.T. Hack), pp. 279-297. Unwin Hyman, Winchester, Massachusetts.

Maxwell, R.W.Jr., Origin and chronology of Alabama River terraces. *Gulf Coast Association of Geological Societies Transactions*, **21**, 83-95.

Mayden, R.L. (1987) Pleistocene glaciation and historical biogeography of North American central-highland fishes. *Kansas Geological Survey Guidebook Series*, **5**, 141-151.

Mayden, R.L. (1988) Vicariance biogeography, parsimony, and evolution in North American freshwater fishes. *Systematic Zoology*, **37**, 329-355.

McKeown, F.A., Jones-Cecil, M., Askew, B.L. & McGrath, M.B. (1988) Analysis of stream-profile data and inferred tectonic activity, eastern Ozark Mountains region. *U.S. Geological Survey Bulletin*, **1807**, 1-39.

Melhorn, W.N. & Kempton, J.P. (1991) The Teays system; a summary. *Geological Society of America Special Paper*, **258**, 125-128.

Mendelson, T.C., Imhoff, V.E. & Venditti, J.J. (2007) The accumulation of reproductive barriers during speciation: postmating barriers in two behaviorally isolated species of darters (Percidae: Etheostoma). *Evolution*, **61**, 2596-2606.

Meylan, M.A. & Li, Z. (1995) Geologic mapping of south-central Mississippi: a model for the distribution of Neogene and Quaternary sediments. *Gulf Coast Association of Geological Societies Transactions*, **45**, 435-440.

Milici, R.C. (1968) Mesozoic and Cenozoic physiographic development of the lower Tennessee River: in terms of the dynamic equilibrium concept. *Journal of Geology*, **76**, 472-479.

Miller, R.R. (2005) *Freshwater fishes of México*. University of Chicago Press, Chicago.

Mills, H.H. & Kaye, J.M. (2001) Drainage history of the Tennessee River: review and new metamorphic quartz gravel locations. *Southeastern Geology*, **40**, 75-97.

Morrone, J.J. (2009) *Evolutionary biogeography*. Columbia University Press, New York.

Near, T.J. & Benard, M.F. (2004) Rapid allopatric speciation in logperch darters (Percidae: Percina). *Evolution*, **58**, 2798-2808.
Near, T.J. & Keck, B.P. (2005) Dispersal, vicariance, and timing of diversification in *Nothonotus* darters. *Molecular Ecology*, **14**, 3485-3496.

Near, T.J., Bolnick, D.I. & Wainwright, P.C. (2005) Fossil calibrations and molecular divergence time estimates in centrarchid fishes (Teleostei: Centrarchidae). *Evolution*, **59**, 1768-1782.

Near, T.J., Kassler, T.W., Koppelman, J.B., Dillman, C.B. & Phillipp, D.P. (2003) Speciation in North American black basses, *Micropterus* (Actinopterygii: Centrarchidae). *Evolution*, **57**, 1610-1621.

Near, T.J., Bossu, C.M., Bradburd, G.S., Carlson, R.L., Harrington, R.C., Hollingsworth, P.R.Jr., Keck, B.P. & Etnier, D.A. (2011) Phylogeny and temporal diversification of darters (Percidae: Etheostomatinae). *Systematic Biology*, **60**, 565-595.

Nelson, G. & Ladiges, P.Y. (1992) Information content and fractional weight of three-item statements. *Systematic Biology*, **41**, 490-494.

Nelson, G. & Ladiges, P.Y. (1996) Paralogy in cladistic biogeography and analysis of paralogy-free subtrees. *American Museum Novitates*, **3167**, 1-58.

Niemiller, M.L., Fitzpatrick, B.M., Shah, P., Schmitz, L. & Near, T.J. (2012) Evidence for repeated loss of selective constraint in rhodopsin of amblyopsid cavefishes (Teleostei: Amblyopsidae). *Evolution*, **67**, 732-748.

Opdyke, N.D., Spangler, D.P., Smith, D.L., Jones, D.S. & Lindquist, R.C. (1984) Origin of the epeirogenic uplift of Pliocene-Pleistocene beach ridges in Florida and development of the Florida karst. *Geology*, **12**, 226-228.

Otvos, E.G. (1981) Tectonic lineaments of Pliocene and Quaternary shorelines, northeast Gulf Coast. *Geology*, **9**, 398-404.

Otvos, E.G. (1998) Citronelle Formation, Northeastern Gulf Coastal Plain: Pliocene stratigraphic framework and age issues. *Gulf Coast Association of Geological Societies Transactions*, **48**, 321-333.

Otvos, E.G. (2005) Numerical chronology of Pleistocene coastal plain and valley development; extensive aggradation during glacial low sea-levels. *Quaternary International*, **135**, 91-113.

Otvos, E.G. (2009) Pleistocene Gulf Coastal Plain: regional stratigraphic and geomorphic contrasts. Intensive erosion related to sea-level decline and hinterland uplift. *Gulf Coast Association of Geological Societies Transactions*, **59**, 585-600.
Page, L.M. & Burr, B.M. (2011) *Peterson field guide to freshwater fishes of North America north of Mexico*, 2nd edition.

Houghton Mifflin Harcourt, Boston.

Parenti, L.R. & Ebach, M.C. (2009) *Comparative biogeography*. University of California Press, Berkeley.

Parenti, L.R. & Ebach, M.C. (2013) Evidence and hypothesis in biogeography. *Journal of Biogeography*, **40**, 8013-820.

Pease, P.P. & Gomez, B. (1997) Landscape development as indicated by basin morphology and the magnetic polarity of cave sediments, Crawford Upland, south-central Indiana. *American Journal of Science*, **297**, 842-858.

Pflieger, W.L. (1971) A distributional study of Missouri fishes. *University of Kansas Publications, Museum of Natural History*, **20**, 225-570.

Phillips, J.D. (2009) Avulsion regimes in southeast Texas rivers. *Earth Surface Processes and Landforms*, **34**, 75-87.

Potter, P.E. (1955) The petrology and origin of the Lafayette Gravel part 2. Geomorphic History. *Journal of Geology*, **63**, 115-132.

Pramuk, J.B., Grose, M.J., Clarke, A.L., Greenbaum, E., Bonaccorso, E., Guayasamin, J.M., Smith-Pardo, A.H., Benz, B.W., Harris, B.R., Siegfried, E., Reid, Y.R., Holcroft-Benson, N. & Wiley, E.O. (2007) Phylogeny of finescale shiners of the genus *Lythrurus* (Cypriniformes: Cyprinidae) inferred from four mitochondrial genes. *Molecular Phylogenetics and Evolution*, **42**, 287-297.

Price, R.C. & Whetstone, K.N. (1977) Lateral stream migration as evidence for regional geologic structures in the eastern Gulf Coastal Plain. *Southeastern Geology*, **18**, 129-147.

Prince, P.S. & Spotila, J.A. (2013) Evidence of transient topographic disequilibrium in a landward passive margin river system: knickpoints and paleo-landscapes of the New River basin, southern Appalachians. *Earth Surface Processes and Landforms*, in press.

Prince, P.S., Spotila, J.A. & Henika, W.S. (2011) Stream capture as driver of transient landscape evolution in a tectonically quiescent setting. *Geology*, **39**, 823-826.

Ricklefs, R.E. & Bermingham, E. (1999) Taxon cycles in the Lesser Antillean avifauna. *Ostrich*, **70**, 49-59.

Ricklefs, R.E. & Cox, G.W. (1972) Taxon cycles in the West Indian avifauna. *American Naturalist*, **106**, 195-219.

Ricklefs, R.E. & Cox, G.W. (1978) Stage of taxon cycle, habitat distribution, and population density in the avifauna of the west indies. *American Naturalist*, **112**, 875-895.
Roe, K.J., Mayden, R.L. & Harris, P.M. (2008) Systematics and zoogeography of the Rock Basses (Centrarchidae: Ambloplites). *Copeia*, **2008**, 858-867.

Rosen, D.E. (1978) Vicariant patterns and historical explanation in biogeography. *Systematic Zoology*, **27**, 159-188.

Rubey, W.W. (1952) Geology and mineral resources of the Hardin and Brussels Quadrangles (in Illinois). *U.S. Geological Survey Professional Paper*, **218**, 1-179.

Saucier, R.T. (1994) *Geomorphology and quaternary geologic history of the lower Mississippi Valley*. U.S. Army Corps of Engineers, Mississippi River Commission, Vicksburg.

Schönhuth, S. & Mayden, R.L. (2010) Phylogenetic relationships in the genus *Cyprinella* (Actinopterygii: Cyprinidae) based on mitochondrial and nuclear gene sequences. *Molecular Phylogenetics and Evolution*, **55**, 77-98.

Schönhuth, S., Blum, M.J., Lozano-Vilano, L., Neely, D.A., Varela-Romero, A., Espinosa, H., Perdices, A. & Mayden, R.L. (2011) Inter-basin exchange and repeated headwater capture across the Sierra Madre Occidental inferred from the phylogeography of Mexican stonerollers. *Journal of Biogeography*, **38**, 1406-1421.

Self, R.P. (1986) Depositional environments and gravel distribution in the Plio-Pleistocene Citronelle Formation of southeastern Louisiana. *Gulf Coast Association of Geological Societies Transactions*, **36**, 561-573.

Self, R.P. (2000) The pre-Pliocene course of the lower Tennessee River as deduced from river terrace gravels in southwest Tennessee. *Southeastern Geology*, **39**, 61-70.

Self, R.P. (2002) Petrology of Cretaceous to Quaternary gravels from the western valley of the Tennessee River. *Journal of the Tennessee Academy of Science*, **77**, 52-56.

Sheets, R.A. & Simonson, L.A. (2006) Compilation of regional ground-water divides for principal aquifers corresponding to the Great Lakes Basin, United States. *U.S. Geological Survey Scientific Investigations Report*, **2006-5102**, 1-23.

Staheli, A.C. (1976) Topographic expression of superimposed drainage on the Georgia Piedmont. *Geological Society of America Bulletin*, **87**, 450-452.

Stanford, S.D. (2010) Onshore record of Hudson River drainage to the continental shelf from the late Miocene through the late Wisonsinan deglaciation, USA: synthesis and revision. *Boreas*, **39**, 1-17.
Stanford, S.D., Ashley, G.M., Russell, E.W.B. & Brenner, G.J. (2002) Rates and patterns of late Cenozoic denudation in the northernmost Atlantic Coastal Plain and Piedmont. *Geological Society of America Bulletin*, **114**, 1422-1437.

Starnes, W.C. & Etnier, D.A. (1986) Drainage evolution and fish biogeography of the Tennessee and Cumberland rivers drainage realm. *The zoogeography of North American freshwater fishes* (ed. by C.H. Hocutt & E.O. Wiley), pp. 325-361.

John Wiley & Sons, New York.

Stefferud, J.A., Gido, K.B. & Propst, D.L. (2011) Spatially variable response of native fish assemblages to discharge, predators, and habitat characteristics in an arid-land river. *Freshwater Biology*, **56**, 1403-1416.

Strain, W.S. (1970) Late Cenozoic bolson integration in the Chihuahua tectonic belt. *The geologic framework of the Chihuahua tectonic belt* (ed. by K. Seewald & D. Sundeen), pp. 56-59. West Texas Geological Society & University of Texas at Austin.

Stults, D.Z., Axsmith, B.J. & Yu-Sheng, L. (2010) Evidence of white pine (*Pinus subgenus Strobus*) dominance from the Pliocene northeastern Gulf of Mexico Coastal Plain. *Palaeogeography, Palaeoclimatology, Palaeoecology*, **287**, 95-100.

Swofford, D. L. 2003. PAUP*. *Phylogenetic Analysis Using Parsimony (*and Other Methods*)*. Version 4. Sinauer Associates, Sunderland, Massachusetts.

Taylor, C.M. & Lienesch, P.W. (1995) Environmental correlates of distribution and abundance for *Lythrurus snelsoni*: a range-wide analysis of an endemic fish species. *Southwestern Naturalist*, **40**, 373-378.

Taylor, C.M. & Lienesch, P.W. (1996) Regional parapatry of the congeneric cyprinids *Lythrurus snelsoni* and *L. umbratilis*: species replacement along a complex environmental gradient. *Copeia*, **1996**, 493-497.

Teller, J.T. & Goldthwait, R.P. (1991) The old Kentucky River; a major tributary to the Teays River. *Geological Society of America Special Paper*, **258**, 29-41.

Thompson, H.D. (1939) Drainage evolution in the southern Appalachians. *Bulletin of the Geological Society of America*, **50**, 1323-1356.

Thompson, H.D. (1949) Drainage evolution in the Appalachians of Pennsylvania. *Annals of the New York Academy of Sciences*, **52**, 33-62.

Todd, J.E. (1917) History of Kaw Lake. *Transactions of the Kansas Academy of Science*, **28**, 187-199.
Torak, L.J., Painter, J.A. & Peck, M.F. (2010) Geohydrology of the Aucilla-Suwannee-Ochlockonee river basin, south-central Georgia and adjacent parts of Florida. *U.S. Geological Survey Scientific Investigations Report, 2010-5072*, 1-78.

Upchurch, P. & Hunn, C.A. (2002) "Time": the neglected dimension in cladistic biogeography? Geobios, Mémoire spécial 24, 277-286.

Van Arsdale, R., Bresnahan, R., McCallister, N. & Waldron, B. (2007) Upland complex of the central Mississippi River valley: its origin, denudation, and possible role in reactivation of the New Madrid seismic zone. *Geological Society of America Special Paper, 425*, 177-192.

Viele, G.W. & Thomas, W.A. (1989) Tectonic synthesis of the Ouachita orogenic belt. *The Appalachian-Ouachita orogen in the United States* (ed. by R.D. Hatcher, Jr., W.A. Thomas, & G.W. Viele), pp. 695-728. Geological Society of America, Boulder, Colorado.

Walters, D.M., Leigh, D.S., Freeman, M.C., Freeman, B.J. & Pringle, C.M. (2003) Geomorphology and fish assemblages in a Piedmont river basin, U.S.A. *Freshwater Biology, 48*, 1950-1970.

Walters, D.M., Blum, M.J., Rashleigh, B., Freeman, B.J., Porter, B.A. & Burkhead, N.M. (2008) Red shiner invasion and hybridization with blacktail shiner in the upper Coosa River, USA. *Biological Invasions, 10*, 1229-1242.

Wayne, W.J., Aber, J.S., Agard, S.S., Bergantino, R.N., Bluemle, J.P., Coates, D.A., Cooley, M.E., Madole, R.F., Martin, J.E., Mears, B.Jr., Morrison, R.B. & Sutherland, W.M. (1991) Quaternary geology of the northern Great Plains.

*Quaternary non-glacial geology* (ed. by R.B. Morrison), pp. 441-476. Geological Society of America, Boulder, Colorado.

Webb, S.A., Graves, J.A., Macias-Garcia, C., Magurran, A.E., Fioghil, D.Ó. & Ritchie, M.G. (2004) Molecular phylogeny of the livebearing Goodeidae (Cyprinodontiformes). *Molecular Phylogenetics and Evolution, 30*, 527-544.

Wilkinson, C.D. & Edds, D.R. (2001) Spatial pattern and environmental correlates of a midwestern stream fish community: including spatial autocorrelation as a factor in community analysis. *American Midland Naturalist, 146*, 271-289.

Williams, D.M. & Ebach, M.C. (2006) The data matrix. *Geodiversitas, 28*, 409-420.

Witzke, B.J. & Ludvigson, G.A. (1990) Petrographic and stratigraphic comparisons of sub-till and inter-till alluvial units in western Iowa: implications for development of the Missouri River drainage. *Midwest Friends of the Pleistocene Field Conference, 36*, 119-143.
Wund, M.A., Baker, J.A., Clancy, B., Golub, J.L. & Foster, S.A. (2008) A test of the "flexible stem" model of evolution: ancestral plasticity, genetic accommodation, and morphological divergence in the threespine stickleback radiation.

*American Naturalist*, **172**, 449-462.

Zaragüeta-Bagils, R. & Bourdon, E. (2007) Three-item analysis: hierarchical representation and treatment of missing and inapplicable data. *Comptes Rendus Palevol*, **6**, 527-534.

Zaragüeta Bagils, R., Ung, V., Grand, A., Vignes-Lebbe, R., Cao, N. & Ducasse, J. (2012) LisBeth: new cladistics for phylogenetics and biogeography. *Comptes Rendus Palevol*, **11**, 563-566.
BIOSKETCHES

**Chris Hoagstrom**, an Associate Professor, is interested in freshwater fish ecology and conservation at all spatiotemporal scales. He is particularly interested in fish community assembly and how fish assemblages and habitat conditions interact.

**Visotheary Ung**, a PhD student, is working on Southeast Asia biogeography. She is interested in methodology and specialty cladistics approach to historical biogeography.

Kathie Taylor...

Author contributions: C.W.H. conceived the ideas and gathered phylogenetic, distributional, and geological evidence; V.R.U. developed and conducted the three-item analysis; K.T. developed and produced the maps; C.W.H. led the writing; all authors provided comments and feedback and approved the final version of the manuscript
<table><caption>**Table 1** Clades of North American fishes used for analysis. Geographical taxa are monophyletic species groups occupying a single highland area (Parenti & Ebach, 2009). References provide phylogenetic and distributional information used in this study. See Appendix S1 for more detail and Miller (2005) and Page & Burr (2011) for more distributional information.</caption><thead><tr><th>Clade</th><th>Description</th><th>Geographical taxa<br>(number)</th><th>References</th></tr></thead><tbody><tr><td>Campostoma</td><td>Cyprinid genus Campostoma</td><td>10</td><td>Blum et al., 2008;<br>Dominguez-Dominguez et al., 2011;<br>Schönhuth et al., 2011<br>M.J. Blum (Tulane University, personal<br>communication, 2012)</td></tr><tr><td>Cyprinella</td><td>Cyprinid genera Codoma, Cyprinella, &amp; Tampichthys</td><td>29</td><td>Schönhuth &amp; Mayden, 2010</td></tr><tr><td>Moxostoma</td><td>M. Iachneri clade, genus Moxostoma</td><td>9</td><td>Jenkins, 1980; Doosey et al., 2010</td></tr><tr><td>Micropterus</td><td>Genus Micropterus</td><td>8</td><td>Near et al., 2003, 2005; Bagley et al., 2011</td></tr><tr><td>Oligocephalus<br>of Etheostoma</td><td>Percid sub-genera Oligocephalus &amp; Microperca</td><td>31</td><td>Near et al., 2011; Suttkus et al., 2012</td></tr><tr><td>Pileoma</td><td>Sub-genus of Percina</td><td>12</td><td>Near &amp; Benard 2004; Near et al., 2011</td></tr></tbody></table>
<table><caption>**Table 2** Derived members of fish clades used in this study (Table 1) that are not restricted to rocky, highland-type habitats and are widespread across alluvial plains. These are putative stage 1 and stage 2 species within the taxon cycle (Ricklefs & Cox, 1972; Ricklefs & Bermingham, 1999) and are listed by region.</caption><thead><tr><th>Clade</th><th>Coastal Plains</th><th>Glacial Plains</th><th>Great Plains</th><th>Rio Grande Rift</th></tr></thead><tbody><tr><td>Campostoma</td><td></td><td>C. pullum<br>C. oligolepis</td><td>C. plumbeum</td><td></td></tr><tr><td>Cyprinella</td><td>C. leedsi<br>C. suavis<br>C. venusta</td><td>C. analostana<br>C. lutrensis<br>C. spiloptera<br>C. whipplei</td><td>C. lutrensis</td><td>C. lutrensis</td></tr><tr><td>Moxostoma</td><td>M. poecilurum</td><td>M. duquesnii<br>M. valenciennesi</td><td></td><td></td></tr><tr><td>Micropterus</td><td>M. floridanus<br>M. punctulatus<br>M. salmoides</td><td>M. dolomieu<br>M. salmoides</td><td></td><td></td></tr><tr><td>Oligocephalus</td><td>E. collettei<br>E. proeliare<br>E. swaini group</td><td>E. asprigene<br>E. caeruleum<br>E. exile<br>E. spectabile</td><td>E. pulchellum</td><td></td></tr><tr><td>Pileoma</td><td>P. macrolepida<br>P. suttkusi</td><td>P. caprodes</td><td>P. caprodes</td><td></td></tr></tbody></table>
**Figure 1** Highland areas occupied by endemic highland-fish taxa used in this study. (a) Modern river drainages in association with highland areas. Areas are: 1 Pánuco, 2 Lerma, 3 Parras, 4 Cabeza de Vaca, 5 Conchos, 6 Devils, 7 Capitan, 8 Coahuila, 9 Llano, 10 Ouachita, 11 Sabine, 12 Ozark, 13 Loess, 14 Moraine, 15 Tennessee, 16 Ohio, 17 Mobile, 18 Pine, 19 Apalachicola, 20 Ocala, 21 Georgia, 22 Chesapeake. (b) Late Miocene river drainages based on literature review (in text) and results of this study (Fig. 2). Arrow-headed rivers deposited sediments into the ocean, whereas circle-headed rivers deposited sediments inland. Major boundaries to the study area are labelled. Depositional basins referred to in text include: a Illinois Basin, b Mississippi Embayment, c Appalachian Basin, d Salisbury Embayment, e Southeast Georgia Embayment, f Southwest Georgia Embayment, g Mississippi-Red-Tennessee Delta, h Burgos Basin, i Tampico-Misantla Basin, j Delaware Basin, k Chihuahua Trough, and l Parras Basin.

**Figure 2** Intersection tree showing evolutionary relations among highland areas (Fig 1) based on a three-item analysis of six highland-fish clades (Table 1, Appendix S1). Major nodes are labelled with Arabic numerals, whereas sister-area relations are labelled with Roman numerals.
<figure><img src="image_89.png" /></figure>
<figure><img src="image_90.png" /></figure>
<figure><img src="image_91.png" /></figure>
Appendix S1 Presence (X) or absence (-) of geographical taxa among highland areas. Areas are numbered following Figure 1. Clades and taxonomic ref

<table><thead><tr><th rowspan="2">Taxon</th><th colspan="12">Highland Area</th></tr><tr><th>1</th><th>2</th><th>3</th><th>4</th><th>5</th><th>6</th><th>7</th><th>8</th><th>9</th><th>10</th><th>11</th><th>12</th></tr></thead><tbody><tr><td><strong>Campostoma Clade</strong></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>C. anomalum 'anomalum' clade</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td></tr><tr><td>C. anomalum 'michauxi' clade, C. oligolepis southeastern clade</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td></tr><tr><td>C. anomalum 'plumbeum' clade</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>x</td><td>-</td><td>x</td><td>x</td><td>-</td><td>-</td><td>-</td></tr><tr><td>C. anomalum 'pullum' clade</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>x</td><td>-</td><td>x</td></tr><tr><td>C. anomalum 'spadiceum' or 'griseum' Ouachita Mountain clade</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>x</td><td>-</td><td>-</td></tr><tr><td>C. anomalum Virginian clade</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td></tr><tr><td>C. oligolepis Ozark Plateau clade</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>x</td></tr><tr><td>C. ornatum northern clade</td><td>-</td><td>-</td><td>-</td><td>x</td><td>x</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td></tr><tr><td>C. ornatum southern clade</td><td>-</td><td>-</td><td>x</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td></tr><tr><td>C. pauciradii</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td></tr><tr><td><strong>Cyprinella Clade</strong></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Codoma ornata species complex</td><td>-</td><td>-</td><td>x</td><td>x</td><td>x</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td></tr><tr><td>Cyprinella analostana</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td></tr><tr><td>C. alvarezdelvillari</td><td>-</td><td>-</td><td>x</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td></tr><tr><td>C. bocagrande, C. formosa</td><td>-</td><td>-</td><td>-</td><td>x</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td></tr><tr><td>C. caerulea</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td></tr><tr><td>C. callitaenia</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td></tr><tr><td>C. callisema, C. sp. 'cf. nivea'</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td></tr><tr><td>C. camera</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>x</td></tr><tr><td>C. chloristia</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td></tr><tr><td>C. forlonensis</td><td>x</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td></tr><tr><td>C. galactura</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>x</td></tr><tr><td>C. garmani</td><td>-</td><td>-</td><td>x</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td></tr><tr><td>C. gibbsi, C. trichroistia</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td></tr><tr><td>C. labrosa, C. pyrrhomelas, C. xaenura, C. zanema, C. sp. thinlip chub</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td></tr><tr><td>C. leedsi</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td></tr><tr><td>C. lepida Río Frio clade</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>x</td><td>-</td><td>-</td><td>-</td><td>-</td></tr><tr><td>C. sp. 'cf. lepida' Nueces River clade</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>x</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td></tr><tr><td>C. lutrensis 'lutrensis' clade</td><td>-</td><td>-</td><td>-</td><td>-</td><td>x</td><td>x</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>x</td></tr><tr><td>C. sp. 'cf. lutrensis' Río Conchos clade</td><td>-</td><td>-</td><td>-</td><td>x</td><td>x</td><td>-</td><td>x</td><td>x</td><td>-</td><td>x</td><td>-</td><td>-</td></tr><tr><td>C. lutrensis 'suavis' clade</td><td>-</td><td>-</td><td>-</td><td>-</td><td>x</td><td>-</td><td>x</td><td>x</td><td>-</td><td>x</td><td>-</td><td>-</td></tr><tr><td>C. panarcys</td><td>-</td><td>-</td><td>-</td><td>-</td><td>x</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td></tr><tr><td>C. proserpina</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>x</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td></tr><tr><td>C. rutila, C. xanthicara</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>x</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td></tr><tr><td>C. spiloptera</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>x</td></tr><tr><td>C. venusta western clade</td><td>-</td><td>-</td><td>-</td><td>-</td><td>x</td><td>-</td><td>-</td><td>x</td><td>-</td><td>x</td><td>-</td><td>-</td></tr><tr><td>C. venusta southeastern clade</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td></tr></tbody></table>
<table><thead><tr><th>Taxon</th><th>1</th><th>2</th><th>3</th><th>4</th><th>5</th><th>6</th><th>7</th><th>8</th><th>9</th><th>10</th><th>11</th><th>12</th></tr></thead><tbody><tr><td>C. whipplei northeastern clade</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>x</td></tr><tr><td>C. whipplei Ouachita Mountain clade</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>x</td><td>-</td><td>-</td></tr><tr><td>Tampichthys species group</td><td>x</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td></tr><tr><td><strong>Moxostoma Clade</strong></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>M. albidum</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>x</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td></tr><tr><td>M. austrinum, M. mascotae</td><td>-</td><td>x</td><td>x</td><td>-</td><td>x</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td></tr><tr><td>M. congestum</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>x</td><td>x</td><td>-</td><td>x</td><td>-</td><td>-</td><td>-</td></tr><tr><td>M. duquesnii northern race</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>x</td><td>x</td><td>-</td></tr><tr><td>M. duquesnii southern race</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td></tr><tr><td>M. sp. 'Apalachicola redhorse'</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td></tr><tr><td>M. poecilurum</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>x</td><td>-</td></tr><tr><td>M. lachneri</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td></tr><tr><td>M. valenciennesi</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td></tr><tr><td><strong>Micropterus Clade</strong></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>M. cataractae</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td></tr><tr><td>M. coosae</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td></tr><tr><td>M. dolomieu</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>x</td><td>-</td></tr><tr><td>M. floridanus</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td></tr><tr><td>M. henshalli, M. punctulatus</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>x</td><td>x</td><td>x</td><td>-</td></tr><tr><td>M. notius</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td></tr><tr><td>M. salmoides</td><td>-</td><td>-</td><td>-</td><td>-</td><td>x</td><td>x</td><td>x</td><td>x</td><td>x</td><td>x</td><td>x</td><td>-</td></tr><tr><td>M. treculii</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>x</td><td>-</td><td>-</td><td>-</td><td>-</td></tr><tr><td><strong>Pileoma Clade</strong></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Percina austroperca</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td></tr><tr><td>P. bimaculata</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td></tr><tr><td>P. burtoni</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>x</td><td>-</td><td>-</td></tr><tr><td>P. caprodes 'caprodes' clade</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td></tr><tr><td>P. caprodes 'semifasciata' Driftless Area clade</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>x</td><td>-</td><td>-</td><td>-</td></tr><tr><td>P. carbonaria</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>x</td><td>-</td><td>-</td><td>-</td></tr><tr><td>P. fulvitaenia</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>x</td><td>x</td><td>-</td></tr><tr><td>P. jenkinsi</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td></tr><tr><td>P. kathae</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td></tr><tr><td>P. macrolepida</td><td>-</td><td>-</td><td>-</td><td>-</td><td>x</td><td>x</td><td>-</td><td>x</td><td>-</td><td>x</td><td>-</td><td>-</td></tr><tr><td>P. rex</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td></tr><tr><td>P. suttkusi</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td></tr><tr><td><strong>Oligocephalus Clade</strong></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Etheostoma artesiae</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>x</td><td>x</td><td>-</td><td>-</td></tr><tr><td>E. asprigene</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>x</td><td>-</td><td>-</td><td>-</td></tr><tr><td>E. australe</td><td>-</td><td>-</td><td>-</td><td>x</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td></tr><tr><td>E. bison, E. cf. spectabile Mamequit darter</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td></tr></tbody></table>
<table><thead><tr><th>Taxon</th><th>1</th><th>2</th><th>3</th><th>4</th><th>5</th><th>6</th><th>7</th><th>8</th><th>9</th><th>10</th><th>11</th><th>12</th></tr></thead><tbody><tr><td>E. burri</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>x</td></tr><tr><td>E. caeruleum northeastern clade</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td></tr><tr><td>E. caeruleum southwestern clade</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>x</td></tr><tr><td>E. collettei, E. ditrema, E. nuchale, E. swaini clade</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>x</td><td>-</td><td>-</td></tr><tr><td>E. cyanorum, E. paludosum</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>x</td><td>-</td><td>-</td></tr><tr><td>E. exile</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td></tr><tr><td>E. fonticola</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>x</td><td>-</td><td>-</td><td>-</td></tr><tr><td>E. fragi, E. uniporum</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>x</td></tr><tr><td>E. grahami</td><td>-</td><td>-</td><td>-</td><td>-</td><td>x</td><td>-</td><td>x</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td></tr><tr><td>E. kantuckeense, E. lawrencei, E. cf. spectabile lhiyo darter, E. tecumsehi</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td></tr><tr><td>E. lepidum</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>x</td><td>-</td><td>x</td><td>-</td><td>-</td><td>-</td></tr><tr><td>E. lugoi, E. segrex</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>x</td><td>-</td><td>-</td><td>-</td><td>-</td></tr><tr><td>E. luteovinctum</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td></tr><tr><td>E. microperca</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>x</td><td>x</td><td>-</td><td>-</td></tr><tr><td>E. proeliare</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>x</td><td>x</td><td>-</td><td>-</td></tr><tr><td>E. pulchellum</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>x</td><td>x</td><td>-</td><td>x</td><td>-</td></tr><tr><td>E. radiosum, E. whipplei</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>x</td><td>-</td><td>x</td><td>-</td></tr><tr><td>E. spectabile 'spectabile' clade</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td></tr><tr><td>E. cf. spectabile Ozark darter</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>x</td><td>-</td></tr><tr><td>E. cf. spectabile Sheltowee darter</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td></tr><tr><td>E. cf. spectabile Wildcat darter</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>x</td><td>-</td></tr><tr><td>E. squamosum</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>x</td><td>-</td></tr><tr><td>E. thompsoni</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>x</td><td>-</td><td>-</td></tr><tr><td>E. whipplei</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>x</td><td>-</td></tr></tbody></table>
# Annexe VI : Comments on the International Code of Area Nomenclature (ICAN)

Zaragüeta-Bagils R., Bourdon E., **Ung V.**, Vignes-Lebbe R. et Malécot V. (2009)

*Journal of Biogeography*, **38**: 1617-1619
a useful approach, both to identify possible
null expectations of relationships that differ
significantly from zero and to serve as a
check on those direct relationships that can
be tested against a null expectation of zero.

In conclusion, on average 62% of the
variation in the number of extinctions on
islands could be explained primarily by the
size of the original avifauna if extinction
were random. In our original analysis of the
real data (Karels et al., 2008), all of the
variables explained only 30% of the varia-
tion in the number of extinctions. The
pattern of extinctions of island avifaunas has
considerable non-random structure and this
is truly 'something of interest' and impor-
tance that requires further study.

TIM J. KARELS¹*, F. STEPHEN
DOBSON²,³, HEATHER
S. ZIMBLER-DELORENZO³
AND AMY L. SKIBIEL³

¹Department of Biology, California State
University, 18111 Nordhoff Street,
Northridge, CA 91330, USA, ²Centre
d'Ecologie Functionelle et Evolutive,
UMR 5175 du CNRS, 1919 route de
Mende, F-34293 Montpellier Cedex 5,
France and ³Department of Biological
Sciences, Auburn University, Auburn, AL
36849, USA

*E-mail: karels@csun.edu

ACKNOWLEDGEMENT

We owe T. M. Blackburn thanks for sug-
gesting that we run simulations of ran-
domly generated data to test the efficacy
of our path model. Although we disagree
with his conclusions concerning the use of
path analysis for explaining influences on
extinctions in oceanic avifaunas, we are
edified by the exchange of ideas.

REFERENCES

Arbuckle, J.L. (2007) *Amos 16.0 user's guide*. Amos Development Corporation, Spring House, PA.

Blackburn, T.M., Cassey, P., Duncan, R.P.,
Evans, K.L. & Gaston, K.J. (2004) Avian
extinction and mammalian introductions
on oceanic islands. *Science*, **305**, 1955–
1958.

Blackburn, T.M., Cassey, P., Evans, K.L., Gaston, K.J. & Duncan, R.P. (2009) The biogeography of avian extinctions on oceanic islands revisited. *Journal of Biogeography*, **36**, 1613–1614.

Didham, R.K., Ewers, R.M. & Gemmell, N.J.
(2005) Comment on 'avian extinction

and mammalian introductions on oceanic
islands'. *Science*, **307**, 1411–1412.

Duncan, R. & Forsyth, D. (2006) Modelling population persistence on islands: mammal introductions in the New Zealand archipelago. *Proceedings of the Royal Society B: Biological Sciences*, **273**, 2969–2975.

Escudero, A., Iriondo, J.M., Olano, J.M., Rubio, A. & Somolinos, R.C. (2000) Factors affecting establishment of a gypsophyte: the case of *Lepidium subulatum* (Brassicaceae). *American Journal of Botany*, **87**, 861–871.

Hood, G.M. (2000) PopTools – software for the analysis of ecological models, version 3.0.6. CSIRO. Available at: http://www.cse.csiro.au/poptools (last accessed 28 September 2008).

Karels, T.J., Dobson, F.S., Trevino, H.S. & Skibiel, A.L. (2008) The biogeography of avian extinctions on oceanic islands. *Journal of Biogeography*, **35**, 1106–1111.

Kline, R.B. (2005) *Principles and practice of structural equation modeling*. Guilford Press, New York, NY.

Li, C.C. (1975) Path analysis: a primer.
Boxwood Press, Pacific Grove, CA.

Petraitis, P.S., Dunham, A.E. & Niewiarowski, P.H. (1996) Inferring multiple causality: the limitations of path analysis. *Functional Ecology*, **10**, 421–431.

Steadman, D. & Martin, P.S. (2003) The late Quaternary extinction and future resumption of birds on Pacific islands. *Earth Science Reviews*, **61**, 133–147.

Trevino, H.S., Skibiel, A.L., Karels, T.J. & Dobson, F.S. (2008) Importance of causal analysis of threats to oceanic avifaunas: reply to Blackburn *et al*. *Conservation Biology*, **22**, 495–497.

Editor: Lisa Manne

doi:10.1111/j.1365-2699.2009.02129.x

On the International Code of
Area Nomenclature (ICAN)

ABSTRACT

A recent work proposed a new naming sys-
tem for biogeographical areas: the Interna-
tional Code of Area Nomenclature (ICAN).
This system aims to standardize area names
used in biogeography and constitutes a key
step towards maturity of this discipline. We
point out a number of issues in this first
version of ICAN. These include the lack

of distinction between classification and
nomenclature, the use of ranks, the criteria
of area definition, the conflation of diagnosis
and description, the ambiguous use of valid/
available, and the system of amendments.
We suggest postponing the initial date of
application of this code until consistent rules
are provided.

**Keywords** Amendment, area definition, biogeography, classification, description, diagnosis, ICAN, nomenclature, rank, type-locality.

Ebach *et al.* (2008) recently proposed a new naming system for biogeographical areas: the International Code of Area Nomenclature (ICAN). This system aims to standardize area names used in biogeography and constitutes a key step towards maturity of this discipline. However, if ICAN's foundations lack precision, ICAN will actually complicate the naming of areas. We would like to raise some critical observations that will contribute to the development of an internally consistent ICAN.

CLASSIFICATION VS.
NOMENCLATURE

First, taxonomic nomenclature is ruled by five codes, not three (as ICAN stipulates). In addition to codes cited by Ebach *et al.* (2008), one can mention the International Code of Nomenclature for Cultivated Plants (Brickell *et al.*, 2004) and the International Code of Virus Classification and Nomenclature (ICVCN) (in Fauquet *et al.*, 2005).

Like the Phylocode, ICAN does not dis-
tinguish between nomenclature and classi-
fication. In their introduction, Ebach *et al.*
(2008) state that '[n]omenclatural rules have
been constantly revised during the 250 years
since Linnaeus first proposed his classifica-
tion system'. Despite the fact that nomen-
clature has been revised since Linnaeus
(1736) proposed his nomenclatural rules,
current nomenclatural codes date mainly
from the 19th century (Strickland *et al.*,
1843; Anonymous, 1847; de Candolle,
1867). A nomenclatural code is not intended
to construct classifications, but is used to
name entities. In other words, nomencla-
tural rules concern names, not taxa or areas.
ICAN Sec. A Art. 3.2 states that 'ICAN does
not endorse any particular method among
those in use for validating or classifying
areas'. Such a statement is trivial. Likewise
in Sec. C Art. 1.1, it is stated that '[a]rea
names may be grouped under more inclu-
sive area names in order to represent a
biogeographical taxonomic hierarchy'. The name 'North America', however, does not contain the name 'New York'. The concept of hierarchy applies to areas, not to names.

A nomenclatural code aims to create objective links between names and concepts (taxa, taxonomic assemblages, temporal intervals or areas) through an object, i.e. a particular specimen called a holotype, whose properties are constant through time. In contrast, the concept associated with a name is subject to scientific discussion. To the greatest extent possible, the link between a scientific concept and the name that refers to that concept must be kept devoid of subjectivity. Similarly, a nomenclatural code must also be normative: decisions taken following its rules must be subject to as little interpretation as possible. Nomenclature should deal with: (1) the objective link between a name and the real world through an object, i.e. the holotype – or, more properly, the onomatophore (the name bearer); (2) synonymy, homonymy and the principle of priority; and (3) definition of publication, validity and availability of names. In its current form, the ICAN draft fails to specify some of these points.

## RANKS

In ICAN, the use of ranks is quite arbitrary, because there may be many more areas (nodes) than ranks, at least in some methods such as area cladistics and Brooks parsimony analysis.

It is not stated in ICAN how higher-rank areas inherit the type-locality of names given to included areas. In a ranked taxonomy, the onomatophore of a species is a holotype, the onomatophore of a genus is its type species and that of a family is its type genus. This system guarantees the link between the name of any supraspecific taxon and the holotype without multiplying onomatophores. In ICAN, the type-locality of a higher-rank area can be different from those of included areas. This renders decisions about synonymy/homonymy virtually impossible.

The statement that '[a] rank can only have one valid name' (Sec. C Art. 2.7) is meaningless. ICAN's authors probably meant 'an area can have only one valid name at a given rank'.

## AREA DEFINITION

Ebach *et al.* (2008) state that areas of endemism 'have been recognized as the principal units of modern biogeographical analyses'. Each one is viewed as 'the area

delimited by the distribution range of a taxon'. ICAN, however, specifies that only a type-locality and diagnosis or description is needed to define a biogeographical area (Sec. C Art. 2.1). Examples given in the introduction only include geographical descriptions and do not take taxa into account. This contradicts the statement that '[t]he Northern Adriatic would be synonymized with the Central Adriatic, for example, if it were concluded that the presumed endemics live in both the Central and Northern Adriatic'. In this case, taxonomic distributions are used to describe the Adriatic, not geography. In addition, it is uncertain whether the northern Adriatic and central Adriatic would be synonymized if only some endemics were found in both areas. ICAN's authors should clarify their criteria, because the consistency of ICAN depends on the rigour of area definition.

Further, we did not find in ICAN any reference to the temporal significance of named areas. For instance, Ebach *et al.* (2008) describe the Adriatic by the presence of the Foci del Po. Yet we do not know where the Foci del Po was placed in the Eocene. We suggest that an area name should be associated with an explicit temporal interval (e.g. Recent/Pleistocene Foci del Po) because current biogeographical studies cannot deal with geographical variations through time, and limit themselves to a single geological age. This will prevent synonymization of area names based on studies concerning organisms of different geological ages.

## DIAGNOSIS VS. DESCRIPTION

Ebach *et al.* (2008) conflate diagnosis and description throughout the ICAN draft (including Sec. C Art 2). For instance, they state that 'the name and type-locality must be linked to a diagnosis (ICAN Sec. C Art. 2.3), which may consist of a detailed description of the area'. However, ICAN (Sec. C Art 4.2) states that '[a] name can be rejected if it has the same diagnosis, description, geographical coordinate or distribution as an existing name'. The latter article distinguishes these two concepts without means of comparing them, i.e. without explaining in which case a description may be identical to a diagnosis. According to the Botanical (McNeill *et al.*, 2006; art. 32.2) and the Zoological (International Commission on Zoological Nomenclature, 1999) Codes, a diagnosis is a statement of the characters that distinguish a taxon from other taxa. In contrast, a

description is a list of properties of the taxon itself (Vignes, 1991). In the context of ICAN, a diagnosis should compare the extent of the described area with those of published areas, while the 'description' paragraph should include a list of localities and endemic taxa. In reference to ICAN Sec. C Art 4.2 we suggest that decisions about rejection be taken only for diagnosis vs. diagnosis and description vs. description.

## PUBLICATION

ICAN fails to define the date of publication of a name. Ebach *et al.* (2008) state that names have to 'be published in a refereed journal', which implies that monographs or technical/edited books are excluded from the naming system. Sec. C Art. 2.6 states that '[n]ames that are published with a diagnosis and/or descriptions linked to a previous discussion of reference are valid', whereas Sec. C Art. 2.8 states that '[t]he principles of homonymy, synonymy and priority apply to the names within any rank that have valid diagnoses'. Accordingly, it is unclear whether validity applies to names and/or diagnoses. Moreover, Sec. C Art. 2.1 specifies that '[a]n available area name has a type-locality and either a published diagnosis or a description in a refereed publication'. Following this ambiguous statement, an unpublished area name can be available just because a type-locality and a description are published. Likewise, ICAN leaves uncertain which distinction, if any, is made between 'available' and 'valid' throughout the ICAN draft. The two distinct uses of the term valid in the Botanical (valid publication of a name) and Zoological codes (valid name) generate the aforementioned confusion.

## AMENDMENTS

Modifications of ICAN rules are made through a series of amendments (Sec. B Art. 2), instead of being directly incorporated into the code. Thus, independent modifications of an article and its subsequent amendments will eventually be impossible to track. In the future, ICAN will be unusable if this system of amendment is maintained.

## CONCLUSION

In this paper, we point out a number of inconsistencies and ambiguous statements in the first version of ICAN. We suggest
postponing the initial date of application of this code until internally consistent rules are provided. Members of the Systematic and Evolutionary Biogeographical Association are warmly encouraged to provide a completed version of ICAN. Such a universal naming system will significantly increase the efficiency of communication in biogeography and other related disciplines.

RENÉ ZARAGÜETA-BAGILS<fnref n="1" />*,
ESTELLE BOURDON<fnref n="2" />, VISOTHEARY
UNG<fnref n="3" />, RÉGINE VIGNES-LEBBE<fnref n="1" /> AND
VALÉRY MALÉCOT<fnref n="3" />

REFERENCES

Anonymous (1847) The rules of American pomology. *Horticulturist Journal of Rural Art and Rural Taste*, **2**, 273–275.

Brickell, C.D., Baum, B.R., Hetterscheid, W.L.A., Leslie, A.C., Mcneill, J., Trehane, P., Vrugtman, F. & Wiersema, J. (2004) *International Code of Nomenclature for Cultivated Plants*, 7th edn. ISHS, Leuven.

de Candolle, A.P. (1867) *Lois de la nomenclature botanique adoptees par le Congrès International de Botanique*. H. Georg, Geneva.

Ebach, M.C., Morrone, J.J., Parenti, L.R. & Viloria, A.L. (2008) International Code of Area Nomenclature. *Journal of Biogeography*, **35**, 1153–1157.

Fauquet, C.M., Mayo, M.A., Maniloff, J., Desselberger, U. & Ball, L.A. (2005) *Virus taxonomy*. Classification and nomenclature of viruses. 8th Report of the ICTV. Elsevier, San Diego.

International Commission on Zoological Nomenclature (1999) *International Code of Zoological Nomenclature*. The International Trust for Zoological Nomenclature, London.

Linnaeus, C.V. (1736) *Fundamenta botanica*.
S. Schouten, Amsterdam.

McNeill, J., Barrie, F.R., Burdet, H.M., Demoulin, V., Hawksworth, D.L., Marhold, K., Nicolson, D.H., Prado, J., Silva, P.C., Skog, J.E., Wiersema, J.H. & Turland, N.J. (2006) *International Code of Botanical Nomenclature (Vienna code)*. A.R.G. Gantner, Verlag KG.

Strickland, H.E., Philipps, J., Richardson,
J., Owen, R., Jenyns, L., Broderip,

W.J., Henslow, J.S., Shuckard, W.E.,
Waterhouse, G.R., Yarrell, W., Darwin,
C. & Westwood, J.O. (1843) Report of a
committee appointed 'to consider the
rules by which the Nomenclature of
Zoology may be established on a uniform
and permanent basis'. *Report of the 12th
Meeting of the British Association for the
Advancement of Science (Manchester 1842)*
pp. 105-121. J. Murray, London.

Vignes, R. (1991) *Caractérisation automatique de groupes biologiques*. Unpublished PhD Thesis, Université Pierre et Marie Curie, Paris.

Editor: David Bellwood

doi:10.1111/j.1365-2699.2009.02106.x

On the International Code of
Area Nomenclature (ICAN): a
reply to Zaragüeta-Bagils *et al.*

ABSTRACT

In 2007 the Systematic and Evolutionary Biogeographical Association (SEBA) wrote and ratified the first draft of the International Code of Area Nomenclature (ICAN), which was posted subsequently on the SEBA website. The ICAN was published, along with an explanatory discussion, by Ebach *et al*. (Journal of Biogeography, 2008, **35**, 1153–1157), an article that is the subject of criticism by Zaragüeta-Bagils *et al*. (Journal of Biogeography, 2009, **36**, 1617–1619). We welcome discussion of the issues raised by these authors and respond to them briefly here. For many reasons, we reject the proposition that implementation of the ICAN be postponed until it is flawless. The ICAN has already been implemented. Further, it is the nature of nomenclatural codes to be proposed and then revised periodically to suit our applications. Most importantly, standardization of area names in biogeography is long overdue.

Keywords Area classification, area names, area nomenclature, area ranks, ICAN, standards in biogeography.

The first draft of the International Code of Area Nomenclature (ICAN) was written and ratified by members of the Systematic and Evolutionary Biogeographical Association (SEBA) and posted on its website (Ebach

et al., 2007). An explanatory discussion and the text of the ICAN were published subsequently as a Guest Editorial in the *Journal of Biogeography* (Ebach et al., 2008). That editorial was the subject of a critique by Zaragüeta-Bagils et al. (2009) to which we reply briefly here. We are grateful for the attention paid by these authors to the ICAN, particularly by their comment that it ...constitutes a key step toward maturity of [biogeography]'. We offer some comments in anticipation of the benefits of public debate. Our subject headings mirror those in Zaragüeta-Bagils et al.'s (2009) critique.

CLASSIFICATION VERSUS
NOMENCLATURE

The explanatory discussion and the ICAN in Ebach *et al.* (2008) are explicit about the relationship between classification and nomenclature: the ICAN is concerned with nomenclature, not with particular methods to construct biogeographical classifications. That we mentioned the nomenclatural rules and classification of Linnaeus in the same sentence of the explanatory discussion does not mean that we equate or confuse these concepts (Zaragüeta-Bagils *et al.*, 2009). This is one example of how Zaragüeta-Bagils *et al.* (2009) unfortunately conflate the explanatory discussion accompanying the ICAN and the ICAN itself. Similarly, they state that the 'ICAN stipulates' (Zaragüeta-Bagils *et al.*, 2009) that taxonomic nomenclature is ruled by three codes. The three nomenclatural codes for natural living organisms – not those cultivated or viral – are noted in the explanatory discussion, not the ICAN.

Zaragüeta-Bagils et al.'s (2009) rejection of the ICAN statement that '[a]rea names may be grouped under more inclusive area names in order to represent a biogeographical taxonomic hierarchy' using the argument that a hierarchy applies to areas, not names, is beside the point. A hierarchy of areas may be represented by words, by a complex map, or, ideally, both.

RANKS

We agree that the use of classificatory ranks
is arbitrary and gave several examples in our
explanatory discussion of how in practice
these may be applied or even ignored. The
ICAN is criticized because '...the type-
locality of a higher-rank area can be differ-
ent from those of included areas', and
therefore, in the opinion of Zaragüeta-Bagils
et al. (2009), it is impossible to make

<footnote n="1">¹UPMC Université Paris 06, Paléobio-
diversité et Paléoenvironnements,</footnote>

<footnote n="2">²Collège de France, Mécanismes Adaptatifs, UPMC Université Paris 06, Paris and</footnote>

<footnote n="3">³AGROCAMPUS OUEST - Centre d'Angers, Génétique et Horticulture, INRA, Université d'Angers, Angers, France</footnote>

<footnote>*E-mail: rene.zaragueta bagils@upmc.fr</footnote>
# Annexe VII : Xper²: introducing e-Taxonomy

Ung V., Dubus G., Zaragüeta-Bagils R. & Vignes-Lebbe R. (2010)

Bioinformatics, 26 (5) 703-704.
# Xper²: introducing e-taxonomy

Visotheary Ung*, Guillaume Dubus, René Zaragüeta-Bagils and Régine Vignes-Lebbe
UPMC, Université Pierre et Marie Curie, UMR 7207 (CR2P), Centre de Recherche sur la Paléobiodiversité et les Paléoenvironnements, Paris, France

Associate Editor: Alex Bateman

## ABSTRACT

**Motivation:** Computer Aided Identification systems provide users with the resources to relate morpho-anatomic observations with taxa names and to subsequently access other knowledge about the organisms. They have the ability to manage descriptive data and make identifications through interactive keys. They are essential for both authors and users of biodiversity information. Xper² version 2.0 is one of the most user-friendly tools in its category and provides a complete environment dedicated to taxonomic management.

**Availability:** Xper² software can be freely downloaded at http://lis-upmc.snv.jussieu.fr/lis/?q=en/resources/softwares/xper2

**Contact:** visotheary.riviere-ung@snv.jussieu.fr

Received on October 15, 2009; revised on November 24, 2009; accepted on December 28, 2009

## 1 INTRODUCTION

Linnaeus wrote that naming is the first stage of knowledge. Indeed, names of taxa are the main access keys to biological information (Polaszek, 2005). They link the real world of living organisms and the conceptual world proposed by systematists through the description and characterization of taxa. Identifying life forms is crucial for all activities related to biology and their taxonomic descriptions are more than necessary in the study of Biodiversity (Godfray, 2002; Lebbe and Vignes, 1991, 1998). Systematists inventory, study and structure biological diversity as precisely as possible (Matile et al., 1987). All this information may be summarized into knowledge bases to perform identification of specimens for new inventories and monitoring surveys, phylogenetic analyses, as well as biogeographic and ecological studies. A significant increase of this kind of digitized information should be expected in a near future. The deep social and scientific impact of web 2.0 stimulates sharing of digital data and proposals of 'cybertaxonomy' projects to study global biodiversity and climate changes. The generalization and the integration of knowledge-base management systems into current taxonomic work may be the next revolution in taxonomy. International projects are already opening the way of this new, delocalized taxonomy.

## 2 WHAT IS XPER²?

Xper² is a management system for storage, editing, analysis and on-line distribution of descriptive data. It dynamically creates interactive keys for identifying specimens (Fig. 1). Xper² does

<figure><img src="image_92.png" /><figcaption>Fig. 1. A screenshot of the identification module (Genets and Oyans knowledge base).</figcaption></figure>

not require any special computer skills. It is written in Java and available on Windows™ Mac™ or Linux in French, English or Spanish versions. It has a user-friendly and intuitive interface aimed at professional taxonomists as well as naturalists who merely want to identify specimens using a ready-made application. Xper²'s users are taxonomists, teachers, fauna and flora experts (for biodiversity monitoring or survey) and ecologists. Formalization of knowledge is the main difficulty for taxonomists. How to structure and organize different types of data? How to keep the scientific sources of the data in order to maintain traceability? Xper² is designed to manage this rich and heterogeneous information. Divided into four modules, Xper² allows taxonomists to either access the descriptive environment, the taxonomic description module, the management tools or the free access key:

1. descriptive model: creation and editing of a standardized description (structured into characters, states, groups and logical dependencies);

2. taxonomic editor: characteristics of the taxon, i.e. its name, descriptions, comments and pictures;

3. management tools (see Section 3.4); and

4. free access key: creation of interactive identification keys (IiK).

<footnote>*To whom correspondence should be addressed.</footnote>
# 3 NEW FUNCTIONALITIES

## 3.1 Import/export structural descriptive data

Many international research programs focus on the establishment of standard formats for data and knowledge exchange. Xper² implements the structured descriptive data (Hagedorn et al., 2006), the exchange format for taxonomic descriptions that has emerged from the work of the Biodiversity Information Standards (TDWG, http://www.tdwg.org, formerly known as taxonomic database working group). Xper² can seamless import/export data from/to SDD, following international recommendations.

## 3.2 Nexus export

Xper² extends the descriptive morpho-anatomic work and the taxonomic expertise of taxonomists into phylogenetic studies. A knowledge base can be exported in nexus format and directly be run in most phylogeny programs such as PAUP*.

## 3.3 HTML export

Xper² provides users with a user-friendly tool to instantly format the knowledge base in a set of HTML files according to a defined cascading style sheet provided by the application. These pages include a form for each taxon displaying its complete description, pictures and any additional comments. By this way, monographies can be easily computed and published in order to serve the entire community. As an example, a website on monitor lizards (http://lis-upmc.snv.jussieu.fr/varanID) has been created by a naturalist group from the Paris Museum.

## 3.4 An accurate knowledge representation and more tools to analyze descriptive data

Xper² offers solutions to control and prevent inconsistencies via a check base function. Furthermore, analyzing descriptive data is one of the most important taxonomists' tasks and Xper² gives tools to carry it out:

* Xper² is able to manage uncertainty by selecting several descriptor-states and using logical operators (logical AND, logical XOR, logical NOT).

* Mismatch threshold: one can define a different threshold according to his expertise level when performing identification.

* Instant comparison of all taxa in a matrix summarizing descriptions.

# 4 FORTHCOMING DEVELOPMENTS

Management of extended descriptor types are under development. This includes management of numerical data, hierarchical or structured descriptor states, computed values, or any other user-declared type (Chalubert and Vignes, 2006). Associated tools allowing writing descriptions or diagnoses in natural language or proposing different kinds of similarity index among taxa will be implemented in future versions.

# 5 AVAILABILITY

Xper² is a versatile tool for editing and using taxonomic descriptions. It is free of charge and can be downloaded at http://lis-upmc.snv.jussieu.fr/lis/?q=en/resources/softwares/xper2. A mailing-list facility provides users with any help or advice that may be needed. Authors choose how they want to use or publish their knowledge base: distributed on CD (i.e. Mascarene Corals identification tool, Conruyt et al., 2000) or online publication (e.g. XperBotanica—a project to identify the flora of France, http://lis-upmc.snv.jussieu.fr/xperbotanica).

# 6 CONCLUSION

Many international projects such as GBIF, EDIT, BIOTA, Catalogue of Life, Encyclopedia of Life, Key2Nature, etc. have a common purpose: sharing knowledge and providing free web access to Biodiversity Information (Bisby et al., 2002; Ramsey and Janzen, 2002; Tillier and Roberts, 2006). In this context of collaborative work, Xper² fits with current taxonomists' practices and gathers functionalities to perform a suitable e-taxonomy!

# ACKNOWLEDGEMENTS

The authors thank B. Pavie, C. Gallut and A. Chalubert for their contribution to the initial Xper² version in Java. They thank all the Xper² users and especially Fabienne Audebert, Ona Maiocco, Philippe Gaubert and David Caron for their bases and also members of the EDIT project for their comments on the software. They are also very grateful to Dr Kurt Liittschwager for comments and style improvement.

Funding: Xper² development has been partially funded by the BioInfo 2002 grant of the CNRS and by the French Ministère for Research and New Technology (No. 04L370—Project Xper Botanica, 2005–07).

Conflict of interest: none declared.

# REFERENCES

Bisby, F. et al. (2002) Taxonomy at a click of mouse. *Nature*, **418**, 367.

Chalubert, A. and Vignes, R. (2006) A New Model for Descriptive Knowledge. *Proceedings of TDWG Annual Meeting 2006*. Available at http://www.tdwg.org/proceedings/article/view/32.

Conruyt, N. et al. (2000) IKBS, a knowledge base management system for reengineering systematics: application to corals of the Mascarene Archipelago. *ICRS 2000—Ninth International Conference for Reef Studies*. ICRS, Bali, p. 17.

GBIF, Global Biodiversity Information Facility, http://www.gbi.fv.org.

Godray H.C.J. (2002) Challenges for Taxonomy. *Nature*, **417**, 17–19.

Hagedorn, G. et al. (2006). The Structured Descriptive Data (SDD) w3c-xml-schema, version 1.1.

Lebbe.J. and Vignes.R. (1991) Génération de graphes d'identification à partir de descriptions de concepts. In Kodratoff, Y. and Diday, E. (eds) *Induction Symbolique et Numérique à partir de données*. Cepadaes, France, pp. 193–239.

Lebbe.J. and et Vignes.R. (1998). State of the art in computer-aided identification in biology. *Oecologia*, **24**, 305–317.

Matiile, L. et al. (1987) Introduction à la systématique zoologique. *Biosistema*, 1–67.

Polaszek, A. (2005) A universal register for animal names. *Nature*, **437**, 477.

Ramsey, S. and Janzen, D. (2002) All living things, online. *Nature*, **418**, 262–263.

Tillier, S. and Roberts, D. (2006) Taxonomy on the fly in a European web project. *Nature*, **440**, 24.
<figure><img src="image_93.png" /></figure>
# Annexe VIII : IKEY+ : a new single-access key generation web service.

Burguiere T., Causse F., **Ung V.** et Vignes-Lebbe R. (2013)

Systematique Biology, 62(1): 157-161.
IKey+: A New Single-Access Key Generation Web Service

THOMAS BURGUIERE*, FLORIAN CAUSSE, VISOTHEARY UNG, AND RÉGINE VIGNES-LEBBE

Laboratoire Informatique et Systématique, Museum National d'Histoire Naturelle, Département Histoire de la Terre,
UMR 7207 - C.N.R.S. - M.N.H.N. - U.P.M.C., Paris, France

*Correspondence to be sent to: Thomas Burguiere, Laboratoire Informatique et Systématique, M.N.H.N Département Histoire de la Terre,
Bâtiment de Géologie, CP48, 57 rue Cuvier, 75005 Paris, France,
E-mail: thomas.burguiere@upmc.fr

Thomas Burguiere and Florian Causse contributed equally to this article.

Received 2 April 2012; reviews returned 29 June 2012; accepted 25 July 2012

Associate Editor: David Posada

Abstract.—Single-access keys are a major tool for biologists who need to identify specimens. The construction process of these keys is particularly complex (especially if the input data set is large) so having an automatic single-access key generation tool is essential. As part of the European project VIBRANT, our aim was to develop such a tool as a web service, thus allowing end-users to integrate it directly into their workflow.

IKey+ generates single-access keys on demand, for single users or research institutions. It receives user input data (using the standard SDD format), accepts several key-generation parameters (affecting the key topology and representation), and supports several output formats.

IKey+ is freely available (sources and binary packages) at www identificationkey.fr. Furthermore, it is deployed on our server and can be queried (for testing purposes) through a simple web client also available at www.identificationkey.fr (last accessed 13 August 2012). Finally, a client plugin will be integrated to the Scratchpads biodiversity networking tool (scratchpads.eu). [Systematics; taxonomy; single-access key; web service; biodiversity informatics.]

During the last decade, taxonomy has been going through a revolution towards cybertaxonomy (Wheeler 2008). This is particularly obvious when looking at the numerous initiatives dedicated to the management and monitoring of biodiversity data such as Global Biodiversity Information Facility (GBIF), European Distributed Institute of Taxonomy (EDIT), BIÓTA, Catalogue of Life, Encyclopedia of Life (EOL) or Key To Nature, and now ViBRANT. A significant increase of digitized information should be expected in a near future, and the deep social and scientific impact of web 2.0 stimulates sharing of digital data and proposals of cybertaxonomy projects to study global biodiversity and climate changes. In the current context of distributed knowledge and long-distance collaborations, the European project ViBRANT (http://vbrant.eu, last accessed 13 August 2012) has emerged to bridge the existing gap between tools and services, providing a powerful exemplar platform, the Scratchpads, for the entire community.

ViBRANT is about connecting the people, data and science of
biodiversity (ViBRANT - Objectives)

As studying and monitoring biodiversity remains a strong challenge for biologists, descriptive data management and identification tools are needed to assist them in their daily work. For instance, we developed a complete descriptive data management tool: Xper² (Ung et al. 2010).

Our contribution to ViBRANT is the implementation of an identification key generation web service, because we also have a significant experience in developing key generation tools such as MyKey (Gérard and Vignes-Lebbe 2010). The most common identification keys (described in Hagedorn et al. 2010) are the printed single-access keys published in almost all taxonomic revisionary work. But these printed keys may be difficult to use while collecting samples in the field or when consulting incomplete specimens. Hence, electronic single-access keys, which can be quickly generated with different parameters in order to bypass potentially problematic steps (i.e., steps with characters for which the specimen studied in the field cannot provide information) are a good alternative.

APPROACH

Web Service

There are currently several tools designed to generate single-access keys such as, among others, the Delta software suite (Dallwitz et al. 1993), Pankey (Pankhurst 1988), and MyKey. These tools are well established and produce valid identification keys, but are not without drawbacks. For instance, both Delta and Pankey need to be installed on the end-user's machine and are not cross-platform compatible (Windows, Apple, and Linux). Mykey is a web application, which makes it platform-agnostic, but the end-users cannot submit their own data
<figure><img src="image_94.png" /><figcaption>FIGURE 1. IKey+ use cases.</figcaption></figure>

set to generate a key (the data set has to be manually uploaded by someone in our laboratory). Furthermore, these 3 tools do not use the biodiversity description standard file format, Structured Descriptive Data (SDD) (Hagedorn 2006), for data input.

Our objective, within the ViBRANT project, was to design a free and open-source key generation service using the SDD file format that can be used by independent users or through the ViBRANT OBOE service (also available through Scratchpads). It was thus decided to develop a web service (implementing both the SOAP and REST web service communication protocols), which is particularly suitable for these two use cases (cf. Fig. 1) and allows the client to build complex workflows, with a high level of automation.

## Architecture

Because the main purpose of ViBRANT is to provide a platform that is both open source and easily reusable, we organized IKey+ in two parts:

* an Application Programming Interface (API) consisting of 3 distinct modules; the Data Model, that is, the computer representation of the descriptive data, the Input-Output (IO) module, which parses the SDD input files and loads the data in the Model and generates the output files, and the Algorithm, which uses the data contained in the Model to generate a key and returns it through the IO module.

* a Simple Object Access Protocol (SOAP) or a Representational State Transfer (REST) Service Layer encapsulating the API, which manages the communication between the client and the API.

This allows potential developers to integrate the service into their workflows and adapt it to their specific needs (e.g., integrating the API in a standalone software). The whole application was developed using the J2EE (Java 2, Enterprise Edition) programming environment.

## Web Service Input Parameters

IKey+ has several parameters available to the end-user. These parameters allow the end-user to change the

topology of the key (e.g., pruning the key, promoting some characters), change the visual aspect of the key, or the output format. A complete list of these parameters can be found in the user documentation, available here: http://www.identificationkey.fr/resources/docs/identificationKeyGeneratorWS_UserGuide.pdf (last accessed 13 August 2012).

## Algorithm

The single-access key generation algorithm is a recursive depth-first graph construction algorithm. Its arguments are a list of taxa, *taxaList* and the list of character considered, *charList* (cf. Supplementary Materials, appendices 1, 2 and 3, available at http://datadryad.org, doi:10.5061/dryad.3ft19). The resulting graph (cf. Fig. 2) is a directed acyclic graph, consisting of non-terminal nodes labelled with a character, edges labelled with the states of the character of the previous node, and terminal nodes labelled with taxa. At each step of the algorithm, the best character among those available is selected by the BEST_CHAR function, which iterates over the available characters and returns the character with the greatest discriminant power.

## Discriminant Power of a Character

During the last 50 years, estimating a character's discriminant power has been the central point of key-construction algorithms. Many measurement methods have been suggested, and a review of these methods can be found in Gower and Payne (1975), Pankhurst (1991), and Delgado-Calvo-Flores et al. (2006). In a statistical context, examples include the Bayesian probability and generalized entropy, such as the Shannon entropy used in the ID3 algorithm developed by Quinlan (1986) or the Gini index used in Breiman et al. (1984). In a context where there is no probability associated with the states of the characters, one can use the separation coefficient or the variance as measurement methods of a character's discriminant power.

In IKey+, the discriminant power of a given character *C* is calculated by the DPOWER function and is an estimate of *C*'s ability to differentiate the taxa of the current list. DPOWER is an extension of the Gyllenberg separation factor (Gyllenberg 1963) and
<figure><img src="image_95.png" /><figcaption>FIGURE 2. Formal identification key example.</figcaption></figure>

is a measurement of the number of pairs of taxa discriminated by C, which is a generalization of the variance. Indeed, the variance of a variable can be computed by comparing all pairs of values (here, comparing all pairs of taxa description for C). With different comparison functions, each adapted to a certain type of character (e.g., a categorical or a numerical character), it is possible to obtain a generalized formula to estimate the discriminant power of a given character, even with polymorphic characters or characters with missing data. For categorical characters, DPOWER can use a binary function (boolean comparison) or other functions such as the Sokal and Michener coefficient (Sokal and Michener 1958) or the Jaccard coefficient (Jaccard 1901). For numerical characters (e.g., a size measured in millimetre), the set of values (i.e., all the values entered for C for all taxa) is split in two intervals. In order to determine the threshold value separating these two intervals, we consider the list of min and max value of C for each of the remaining taxa. We then choose from this list the value that separates the remaining taxa into two groups of equal size (±1 taxon). These two intervals are considered as 2 discrete states for the calculation of the discriminant power.

DPOWER iterates over the available taxa (i.e., those that are compatible with the current description), and determines, for each pair of taxa $T_a$ and $T_b$, the dissimilarity (i.e., the possibility of discriminating $T_a$ and $T_b$ with C) that is based on the number of common states of C for $T_a$ and $T_b$ ($n_{11}$), the number of states of C that occur only for $T_a$ ($n_{10}$), the number of states of C that occur only for $T_b$ ($n_{01}$), and the number of states of C that do not occur for neither $T_a$ or $T_b$ ($n_{00}$). The discriminant power is then calculated as

$$DP = \sum_{a} \sum_{b} \text{SCORE}(n_{11}, n_{10}, n_{01}, n_{00}).$$

Three different SCORE measurements are currently available in IKey+, the Xper coefficient (Ung et al. 2010;

Vignes et al. 1989), the Jaccard coefficient, and the Sokal and Michener coefficient.

## BENCHMARKS

### Tests

In order to assess the performance of our web service, we conducted a series of tests using the same input file for every test. This file contains a data set representing the subfamily Cichorieae of the plant family Asteraceae with 303 observable characters, 144 taxa, and their descriptions. This data set was generated and curated as an exemplar group for the EDIT project (Hand et al. 2009) [the file is available here: http://www.infosyslab.fr/vibrant/project/test/Cichorieae-fullSDD.xml (last accessed 13 August 2012)]. The aim of these tests was to evaluate the overall performance of the algorithm, to assess the impact of the various parameters available to the end-user on performance, and to ensure that IKey+ would be robust in a production environment. We measured the time necessary for the web service to respond to 100 identification keys generation queries, while counting the number of rejected queries due to CPU overload (IKey+ rejects any query received when the CPU load of the host server is greater than 80%). We ran one reference test (described in Supplementary Materials, appendix 4, doi:10.5061/dryad.3ft19), and several tests with variations on the input parameters, the web service communication protocol used or the parallelization setups. The complete list of performance test setups is available in Supplementary Materials, appendix 5, doi:10.5061/dryad.3ft19. We also measured the time necessary to generate a single key, using the reference test configuration described in Supplementary Materials, appendix 4, doi:10.5061/dryad.3ft19. Finally, we tested the usability of IKey+ from an end-user perspective, when generating the Cichorieae identification key using the web interface. We asked 11 persons who were not
involved in the development of IKey+ to test the web
service and the web interface. They were given the
SDD formatted Cichorieae data set and were asked to
use the web interface to create the identification key.
We measured the time needed by each test subject to
generate the identification key.

Results

The average length of the paths leading to taxa
that were identified by the algorithm is 4.67 steps,
with the shortest path being 1 step long, and the
longest being 10 steps long. The generation of a single
key took roughly 1.8 s. The results of the other tests
are shown in Supplementary Materials, appendix 6,
doi:10.5061/dryad.3ft19. The reference test took roughly
50 s to complete, that is, 500 ms per query, which is
consistent with the time measured for the generation
of a single key, because the reference test uses 4
simultaneous threads to generate the keys. In this test,
few queries were rejected due to CPU overload. Among
the parameters available to the end-user, only the score
method parameter had a significant influence: when
using the Sokal and Michener score method or the
Jaccard score method, the tests took longer to finish
(~80 s instead of 50 s). The score method parameter had
no impact on the number of rejected queries. Our tests
showed that the communication protocol used (SOAP or
REST) had no influence on the performance of IKey+.

Some taxa do not appear in the generated key, due to
insufficient data in the input file. Indeed, the Cichorieae
data set we used for our tests was created by an
external team. This is because some errors were made
during creation of the Cichorieae data set: some taxa
with unknown data were not specifically marked as
"unknown data", but were left unspecified instead.
Regardless, although these ambiguously coded taxa do
not appear in the resulting identification key by default,
the end-user can choose to have them appear in the key.

When modifying the parallelization of the queries,
we observed significant performance variations. As can
be expected, when launching 100 queries sequentially
(instead of using 4 simultaneous threads launching 25
queries each), the test was 4 times longer, and no queries
were rejected. When we augmented the parallelism
of the queries (e.g., 25 or 100 simultaneous queries),
more queries were rejected (up to 50%). However, when
launching 100 simultaneous queries, with a random
delay at the beginning of each thread, the results (both in
time and number of rejected queries) were comparable to
the performance of the reference test. In the usability test,
the average time needed to generate the identification
key was slightly above 60 s (60.36 s), with the shortest
time measured at 26 s, and the longest time measured
at 121 s. The complete results of the usability test
are available in Supplementary Materials, appendix 7,
doi:10.5061/dryad.3ft19.

DISCUSSION

IKey+ is available and can be installed on any J2EE
application server (e.g., Apache Tomcat). It can generate
single-access keys using a tree or a flat representation
in several output formats (HTML, Wiki, SDD, plain-
text, etc.).

Our test showed that IKey+ performs sufficiently well
to handle a large data set in a relatively short amount of
time and can generate well-optimized key files (average
number of steps to identify a taxon: 4.67). This, combined
with the web service accessibility, makes it possible to
integrate IKey+ in many workflows that might require
a fast and automated key generation process (e.g., a batch
key generation script). Furthermore, an end-user can use
the web interface available at www.identificationkey.fr
to quickly generate a customized identification key,
using the numerous parameters available (affecting the
topology, representation, file format, etc.).

Finally, our tests showed that IKey+ is likely to
be robust in a production environment (i.e., many
simultaneous queries) as it is able to withstand
simultaneous key generation queries (e.g., 4 threads
launching 25 consecutive queries). It is also protected
from cryptic failure, because we implemented a CPU-
load-watching mechanism that automatically rejects a
query (with an explicit error message) whenever the
CPU load exceeds a given threshold (80%). This prevents
a crash of the service, or the generation of incomplete or
corrupt key files.

CONCLUSION

IKey+ is the first key-generation tool available as
a web service with standardized input and output
formats. Our test showed that IKey+ is able to generate
keys rapidly and that it can also be used by an end-user
with the web interface. Finally, the modular and open-
source nature of IKey+ makes it possible for anyone to
reuse its components. For instance, we plan to reuse some
components of the API to develop another web service
that would provide free-access key identification.

LICENSING

As part of the ViBRANT project, IKey+'s source
code is freely available and is licensed under the GNU
General Public License version 2. It is already available
on our google code SVN repository: http://ikey-plus.
googlecode.com/svn/trunk/ (last accessed 13 August
2012). It will be actively maintained by our team for the
next 2 years.

SUPPLEMENTARY MATERIAL

Supplementary material, including Algorithms and
appendices, can be found at http://www.sysbio.
oxfordjournals.org.
## FUNDING

This work was supported by the European Union funded FP7 ViBRANT Project (Contract number RI-261532, Period, December 2010 to November 2013).

## ACKNOWLEDGEMENTS

We sincerely thank Gregor Hagedorn (Julius Kühn Institute, Berlin, Germany) and Andreas Müller (Botanical Garden and Botanical Museum, Berlin, Germany) for sharing their knowledge on the SDD format. We are also grateful to Dave Roberts (Natural History Museum, London, UK) for reviewing an early version of the article and providing style improvements.

## REFERENCES

* **BIOTA.** Available from: URL http://www.edinburgh.ceh.ac.uk/biota/ (last accessed 13 August 2012).

* **Breiman L., Friedman J.H., Olshen R.A.** *Stone C.J.* 1984. Classification and regression trees. Belmont, CA-Wadsworth International Group. Catalogue of Life. Available from: URL http://www.cataloqueoflife.org/ (last accessed 13 August 2012).

* **Dallwitz M.J., Paine T.A., Zurcher E.J.** 1993. User's guide to the delta system: a general system for coding taxonomic descriptions. 4th ed. Available from: URL http://delta-intkey.com (last accessed 13 August 2012).

* **Delgado-Calvo-Flores M., Fajardo-Contreras W., Gibaja-Galindo E.L., Perez-Perez R.** 2006. Xkey: a tool for the generation of identification keys. Expert Syst. Appl. 30:337-351.

* **EDIT.** European Distributed Institute of Taxonomy. Available from: URL http://www.e-taxonomy.eu/ (last accessed 13 August 2012).

* **EOL.** Encyclopaedia of Life. Available from: URL http://eol.org/.

* **GBIF.** Global Biodiversity Information Facility. Available from: URL http://www.gbif.org/ (last accessed 13 August 2012).

* **Gérard D., Vignes-Lebbe R.** 2010. Mykey: a server-side software to create customized decision trees. In: Nimis P.L., Vignes-Lebbe R., editors. Tools for identifying biodiversity: progress and problems. Edizioni Università di Trieste, Trieste, Italy. p. 107-112.

* **Gower J.C., Payne R.W.** 1975. A comparison of different criteria for selecting binary tests in diagnostic keys. *Biometrika* 62:665-672.

* **Gyllenberg H.G.** 1963. A general method for deriving determinative schemes for random collections of microbial isolates. Ann. Acad. Sci. Fenn. Ser. A IV. Biologica 1(69):1-23.

* **Hagedorn G.** 2006. The structured descriptive data (SDD) w3c-xml-schema. Version 1.1 Available from: URL http://wiki.tdwg.org/twiki/bin/view/SDD/Version1dot1 (last accessed 13 August 2012).

* **Hagedorn G., Rambold G., Martellos S.** 2010. Types of identification keys. In: Nimis P.L., Vignes-Lebbe R. editors. Tools for identifying biodiversity: progress and problems. Edizioni Università di Trieste, Trieste, Italy. p. 59-64.

* **Hand R., Kilian N., Raab-Straube E.** 2009. International cichorieae network: Cichorieae portal. Available from: URL http://wp6.cichorieae.e-taxonomy.eu/portal/ (last accessed 13 August 2012).

* **Jaccard P.** 1901. Etude comparative de la distribution florale dans une portion des alpes et des jura. Bull. Soc. Vaud. Sci. Nat. 37:547-579.

* **J2EE.** Java 2, Enterprise Edition. Available from: URL http://www.oracle.com/technetwork/java/javaee/overview/index.html (last accessed 13 August 2012).

* **KeyToNature.** Available from: URL http://www.keytonature.eu/wiki/ (last accessed 13 August 2012).

* **Pankhurst R.J.** 1988. Pankey programs. DELTA Newsletter 1:2.

* **Pankhurst R.J.** 1991. Practical taxonomic computing. Cambridge University Press, Cambridge, UK.

* **Quinlan J.R.** 1986. Induction of decision trees. Mach. Learn. 1:81-106. ISSN 0885-6125. Available from: URL http://dx.doi.org/10.1007/BF00116251 (last accessed 13 August 2012).

* **REST Architecture.** Available from: URL http://www.oracle.com/technetwork/articles/javae/index-137171.html (last accessed 13 August 2012).

* **Scratchpads.** Biodiversity Online. Available from: URL http://scratchpads.eu/ (last accessed 13 August 2012).

* **SOAP.** Simple Object Access Protocol, W3C Recommendation. Version 1.2. Available from: URL http://www.w3.org/TR/soap/ (last accessed 13 August 2012).

* **Sokal R., Michener C.** 1958. A statistical method for evaluating systematic relationships. Univ. Kansas Sci. Bull., (38):1409-1438.

* **Ung V., Dubus G., Zaragüeta-Bagils R., Vignes-Lebbe R.** 2010. Xper2: introducing e-taxonomy. *Bioinformatics* 26(5):703-704.

* **ViBRANTa.** Objectives. Available from: URL http://vbrant.eu/node/1 (last accessed 13 August 2012).

* **ViBRANTb.** Virtual Biodiversity Research and Access Network for Taxonomy. Available from: URL http://vbrant.eu (last accessed 13 August 2012).

* **Vignes R., Lebbe J., Darmoni S.** 1989. Symbolic-numeric approach for biological knowledge representation: a medical example with creation of identification graphs. In E. Diday, editor, Proceedings of the conference on Data analysis, learning symbolic and numeric knowledge. Nova Science Publishers, Inc. Commack, NY, USA. ISBN 0-941743-64-0. p. 389-398.

* **Wheeler, Q.D., editor.** 2008. The new taxonomy. CRC Press Inc. New York, USA.
# Annexe IX : MKey++: Interactive identification webservice

Thomas Burguiere, Antoine Bergamaschi, Florian Causse, **Visotheary Ung**, Nils Paulhe, Frédéric Lison, and Régine Vignes-Lebbe (2013)

En préparation
MKey+: Interactive identification webservice

Thomas Burguiere*†‡, Antoine Bergamaschi‡, Florian Causse, Visotheary Ung, Nils Paulhe, Frédéric Lison, and Régine Vignes-Lebbe

Laboratoire Informatique et Systématique, UMR 7207 – C.N.R.S. - M.N.H.N. - U.P.M.C.

Museum National d'Histoire Naturelle – Département Histoire de la Terre, Paris, FRANCE

May 2013

**Abstract**

Interactive identification keys are a major tool for systematists who need to identify specimens or to transmit their taxonomic expertise to other scientists or amateurs. Our latest software, MKey+, using the SDD standard file format for the input of a descriptive knowledge base and open, standard, web technologies (HTTP, JSON) allows scientists to perform interactive identification online, and brings interactive identification capabilities to any application capable of sending webservice queries.

**keywords:** Systematics, Taxonomy, Interactive identification key, webservice, Biodiversity informatics

<footnote>*to whom correspondence should be addressed</footnote>

<footnote>†thomas.burguiere@upmc.fr</footnote>

<footnote>‡Thomas Burguiere and Antoine Bergamaschi contributed equally to this work</footnote>
Identification keys constitute a major tool for systematist when they need to quickly iden-
tify a specimen, using a reference key. The most common identification keys used by scientists
(described in Hagedorn et al. 2010) are the printed single-access keys published in almost all
taxonomic revisionary work. They consist in a sequence of questions (whose order is fixed by
the author of the key) regarding the morphological characters commonly used to describe a
single or multiple species. This sequence of questions, depending on their answer, lead the
user on a path towards the identification of a specific specimen (cf. example in Figure 1).
These single-access keys, however, are not without drawbacks.

For instance, the sequence of the questions is necessarily a compromise between the discrim-
inant power of the characters (to reduce the number of steps needed to identify a specimen,
the most discriminant characters should appear early in the sequence) and the observation
convenience of the characters (if a character which can't be described for a specific specimen
comes too early in the sequence, the systematist is unable to identify this specimen).

Multiple-access keys provide a good alternative to single-access keys, since they allow the
user to perform an interactive identification i.e. to determine the sequence of questions, so
that the user may skip a question which can't be answered. These keys require a software
component, commonly called a key Player, with which the user chooses the questions order,
answer them, and identifies a specimen. Existing software include the Delta software suite
(Dallwitz et al. 1993, 2013), the Lucid software suite and our own Xper² software (Ung
et al. 2010). All these programs are desktop software, and need to be installed on each
user's machine, prior to any interactive identification. Some (Xper² and Lucid) include a
web applet, which makes it possible to deploy an interactive key on a web server, but the
configuration process is quite cumbersome and the applet technology has substantial security
vulnerabilities. There is currently no way for a systematist to upload his complete knowledge
base and start an interactive identification right away.
<figure><img src="image_96.png" /><figcaption>Figure 1: Comparison of a single-access key (left) and a multiple-access key (right). The single-access key is basically a decision tree and the identification process the traversal of this tree. In the multiple-access key, the identification process consists in the dynamic construction of a traversal path of the potential identification tree.</figcaption></figure>

Within the framework of european project ViBRANT (http://vbrant.eu), which aims at bridging the existing gap between scientists, scientific tools and scientific services, we decided to develop an interactive identification software tool, MKey+. We used the same approach used to develop our single-access key generation software, IKey+ (Burguiere et al. 2013). This approach consists of developing a webservice which provides the main functionalities necessary to perform interactive identification, using the SDD standard format (Hagedorn 2006) for data input, that can be readily queried by HTTP requests to exchange JSON-formatted data (Crockford 2006), then developing a simple web client available online. With this approach, a single user is able to perform an interactive identification using the web client, and software developers can add interactive identification capabilities to their software, provided it is capable of interacting with the webservice using HTTP requests.

## Methods

**Performing an interactive identification using MKey+**. The interactive identification process can be decomposed into 4 steps.
I The user, using any web client, submits the knowledge base to MKey+.

II MKey+ returns the list of the characters which the user can describe, sorted by their discriminant power.

IIIa The user describes a character with the client, and submits this description to MKey+.

IIIb MKey+ returns the list of taxa which are compatible with all the descriptions it has received so far, and the client gets back to step IIIa.

When there is only one remaining taxon, the user has successfully completed his identification.

**Discriminant power of a character.** One of the defining features of the software we develop (Xper², IKey+, and now MKey+) is the calculation of the discriminant power of the characters. This discriminant power is crucial to reduce the number of steps needed to identify a specimen. It indicates the user, at each step of the identification, which of the remaining characters (i.e. the characters that haven't been described yet) are the most discriminant for the remaining taxa (i.e. the taxa that have not been ruled out at a previous step).

There are many methods to calculate the discriminant power of a character, and a review of these methods can be found in Gower and Payne (1975), Pankhurst (1991) and Delgado-Calvo-Flores et al. (2006). In MKey+, the discriminant power of a given character C is an estimate of C's ability to differentiate the taxa of a given list. It is an extension of the Gyllenberg separation factor (Gyllenberg 1963) and is a measurement of the number of pairs of taxa discriminated by C, which is a generalisation of the variance. Indeed, the variance of a variable can be computed by comparing all pairs of values (here, comparing all pairs of taxa description for C). This method is the same as the one we used in IKey+ and Xper², and is thoroughly described in Vignes (1991) and Burguiere et al. (2013).

**Interacting with MKey+**. As stated above, the fact that MKey+ is a webservice makes it possible to interact with in two different ways:
1. a scientist who needs to perform a single identification can use the simple web client we developed <fnref n="1" /> to upload his knowledge base, then start the identification. The only requirements are a modern web browser, an internet connection and an SDD formatted knowledge base.

2. a scientific software developer, without any knowledge of the algorithms involved in the interactive identification process, can write a component which will interact with MKey+ very easily, provided his program is capable of sending HTTP requests. One example of such an integration would be to include an MKey+ client component into the Scratchpads (http://scratchpads.eu, Smith et al. 2011) biodiversity networking tool.

MKey+ is fully functional and is available online, and our web client is available for scientists who want to perform interactive identifications right away. Similarly, software developers can freely add an MKey+ client component to their software, a complete technical description of the identification functions is available (cf. supplementary data). This brings instant full interactive identification online, which we consider to be a significant improvement over existing tools, which at the very least require that users install a key player on their computer. Furthermore, the fact that MKey+ is a webservice makes it possible to consider connecting it to other third-party webservices, which would extend the capabilities of MKey+.

MKey+ is currently capable of handling two kinds of characters: categorical characters which can be described with discrete states (e.g. Shape of the leaf: round / square), and quantitative characters, which can be described with continuous values (e.g. length of the tail: 3cm). Future developments are being considered, to give MKey+ the ability to handle other types of characters, such as color space characters, sequence characters, etc... Finally, we intend to use MKey+ as the interactive identification engine for the next version of our descriptive

<footnote n="1">¹available at http://www.identificationkey.fr/mkey/mkey.html</footnote>
data management software, Xper³.
Visotheary Ung, Guillaume Dubus, René Zaragueta-Bagils, and Régine Vignes-Lebbe.
Xper2: introducing e-taxonomy. *Bioinformatics*, 26(5):703-4, Mar 2010. doi: 10.1093/
bioinformatics/btp715.

Régine Vignes. *Caractérisation automatique de groupes biologiques*. PhD thesis, Université
Pierre et Marie Curie, Apr 1991.
## References

Scratchpads: Biodiversity Online. URL http://scratchpads.eu/.

ViBRANT: Virtual Biodiversity Research and Access Network for Taxonomy. URL http://vbrant.eu.

Thomas Burguiere, Florian Causse, Visotheary Ung, and Régine Vignes-Lebbe. Ikey+: a new single-access key generation web service. *Systematic Biology*, 62(1):157–161, Jan 2013. doi: 10.1093/sysbio/sys069.

D. Crockford. The application/json media type for javascript object notation (json), July 2006. URL http://tools.ietf.org/html/rfc4627.

M.J. Dallwitz, T.A. Paine, and E.J. Zurcher. User's guide to the delta system: a general system for coding taxonomic descriptions. 4th edition, 1993. URL http://delta-intkey.com.

M.J. Dallwitz, T.A. Paine, and E.J. Zurcher. Principles of interactive keys, Jan 2013. URL http://delta-intkey.com/www/interactivekeys.htm.

M. Delgado-Calvo-Flores, W. Fajardo-Contreras, E.L. Gibaja-Galindo, and R. Perez-Perez. Xkey: A tool for the generation of identification keys. *Expert Systems with Applications*, 30:337–351, 2006.

J.C. Gower and R.W. Payne. A comparison of different criteria for selecting binary tests in diagnostic keys. *Biometrika*, 62:665–672, 1975.

H.G. Gyllenberg. A general method for deriving determinative schemes for random collections of microbial isolates. *Ann. Acad. scient. fenn. Ser. A, IV. Biologica*, 1(69):1–23, 1963.

Gregor Hagedorn. The Structured Descriptive Data (SDD) w3c-xml-schema, version 1.1, 2006. URL http://wiki.tdwg.org/twiki/bin/view/SDD/Version1dot1.

Gregor Hagedorn, Gerhard Rambold, and Stefano Martellos. Types of identification keys. In Pier Luigi Nimis and Régine Vignes-Lebbe, editors, *Tools for Identifying Biodiversity: Progress and Problems*, pages 59–64. Edizioni Università di Trieste, 2010.

Richard J. Pankhurst. *Practical taxonomic computing*. Cambridge University Press, 1991. ISBN 0-521-41760-0.

Vincent Smith, Simon Rycroft, Irina Brake, Ben Scott, Edward Baker, Laurence Livermore, Vladimir Blagoderov, and David Roberts. Scratchpads 2.0: a virtual research environment supporting scholarly collaboration, communication and data publication in biodiversity science. *ZooKeys*, 150:53–70, 2011/11/28/ 2011. URL http://dx.doi.org/10.3897/zookeys.150.2193.
<figure><img src="image_97.png" /><figcaption>Figure 1: Comparison of a single-access key identification and a multiple-access key identification.</figcaption></figure>

The single-access key (A) is essentially a decision tree and the identification process is the traversal of this tree.

In the multiple-access key (B), the identification process consists in the dynamic construction of an identification path. A specimen can be identified using different characters, thus creating different identification paths. This is shown in the figure on the right in which both the black and the grey identification paths start from the same character, but depending on the description given by the user, create different identification paths that lead to the same taxon.
<figure><img src="image_98.png" /><figcaption>Figure 2: Interactive identification workflow, using MKey+.</figcaption></figure>

Step I: the client sends the URL of the SDD file containing the dataset on which the identification will be performed. The SDD file is retrieved by the MKey+ *via* the REST Service Layer, its content is parsed and loaded in the Data Model.

Step II: the Dataset content is sent back to the client as a JSON character string, with the characters sorted by their discriminant power in descending order. The Dataset content is also stored in the Dataset cache.

Step IIIa: The specimen is described by the user (*via* the client), i.e. the user describes one character. This description of a single character is sent back to MKey+ *via* the REST Service Layer.

Step IIIb: This description is passed to the identification module, which analyses it, returns the list of remaining taxa contained in the dataset that are compatible with this description, and the list of remaining characters sorted by their new discriminant power. The user goes back to step IIIa, and when only one taxon remains, the identification is complete.
<table><caption>Figure 3: Usability test results</caption><thead><tr><th>User No.</th><th>Time (s)</th><th>Nb Steps</th></tr></thead><tbody><tr><td>1</td><td>145</td><td>7</td></tr><tr><td>2</td><td>125 †</td><td>7</td></tr><tr><td>3</td><td>245</td><td>7</td></tr><tr><td>4</td><td>145</td><td>7</td></tr><tr><td>5</td><td>205</td><td>7</td></tr><tr><td>6</td><td>119</td><td>8</td></tr><tr><td>7</td><td>177</td><td>7</td></tr><tr><td>8</td><td>174</td><td>8</td></tr><tr><td>9</td><td>230</td><td>7</td></tr><tr><td>10</td><td>248 †</td><td>7</td></tr><tr><td>11</td><td>127</td><td>7</td></tr><tr><td>12</td><td>138 †</td><td>7</td></tr><tr><td>Min</td><td>119</td><td>-</td></tr><tr><td>Max</td><td>248</td><td>-</td></tr><tr><td>Mean</td><td>173.17</td><td>-</td></tr><tr><td>Median</td><td>159.50</td><td>-</td></tr><tr><td>SD</td><td>47.89</td><td>-</td></tr></tbody></table>

The times marked by a † symbol indicates users who did not identify the correct specie on their first attempt. The time included in this table is the time they needed to finish their second (and successful) attempt.
RÉSUMÉ

La biogéographie historique est la discipline qui s'occupe d'établir les relations de parenté entre aires biogéographiques, comprises comme des ensembles d'aires de distribution de taxons. L'histoire géologique complexe du Sud Est asiatique et de la région Indo-Pacific en fait une région de choix pour les biogéographes. Nous proposons une nouvelle approche, cladistique, pour l'étude de cette région située autour de la célèbre "Ligne de Wallace", en nous plaçant dans le paradigme de la vicariance où la spéciation apparaît suite à la mise en place d'une barrière géographique (par opposition au modèle dispersaliste à partir d'un centre d'origine). En effet, nous utilisons ici l'analogie existant entre Systématique et Biogéographie pour appliquer des méthodes cladistiques de la Systématique à la Biogéographie. Il s'agit ici de répondre à la question : « *pourquoi existe-t-il une telle distribution des taxons dans cette région du globe ?* » Les résultats attendus sont une meilleure connaissance de l'histoire évolutive de la région ainsi que des traits d'histoire de vie des taxons qui la peuplent. Etant entendu que la distribution conjointe de plusieurs taxons non apparentés ne peut être obtenue par « chance », elle doit forcément rendre compte d'un événement commun (i.e. un événement de vicariance) qui aurait affecté de manière égale ces taxons. L'approche cladistique, que nous proposons n'a été rendue possible que grâce à la finalisation d'un logiciel implémentant la méthode dite d'analyse à 3 éléments (3ia): LisBeth est aujourd'hui disponible en téléchargement librement depuis notre site web et offre de nouvelles perspectives d'analyses à ses utilisateurs, qu'ils soient systématiciens ou biogéographes. Nous montrons que la représentation des caractères offerte par LisBeth autorise de nouvelles inférences quant aux relations de parenté existant entre les aires d'endémismes étudiées. Nous avons ainsi mis en évidence l'émergence d'un « pattern » général de l'analyse conjointe de 35 taxons non apparentés (plantes et animaux).

Dans un deuxième temps nous nous sommes intéressés à une problématique théorique majeure de la biogéographie contemporaine: « *Quid de la dimension temporelle en biogéographie historique ?*». Comment prendre en compte, sans ambiguïté, des échelles temporelles différentes lors d'analyses diachroniques ? Nous proposons ici un modèle, basé sur la 3ia qui permet de considérer la dimension temporelle et de réaliser, de ce fait, de véritables analyses de biogéographie intégrative.

MOTS-CLEFS : Biogéographie historique, biogéographie intégrative, 3ia, Asie du Sud-Est, Pacific Ouest, pattern, process.

ABSTRACT

Historical biogeography is the science that deals with relationships between biogeographic areas, understood as set of taxa distributions. The complex geological history of South East Asia and the Indo-West Pacific region makes it a very interesting area for biogeography studies. We propose a new approach, cladistic, for the study of this area around the famous "Wallace's line", placing our study in the paradigm of vicariance where speciation appears following the implementation of a geographic barrier (as opposed to the dispersal model from a center of origin, classically admitted). Indeed, here we use the existing analogy between Systematics and Biogeography to apply cladistic methods for Systematics to Biogeography. This is to answer the question: "*Why is there such distributions of taxa in this region of the world?*" The expected results are a better understanding of the evolutionary history of the region and the life history traits of taxa that inhabit it. Provided that the joint distribution of several unrelated taxa cannot be obtained by "chance", thus it is necessarily due to a common event (ie a vicariant event) which affected equally these taxa. The cladistic approach we propose has been made possible by the completion of a software that implements the so-called 3-element analysis (3ia) method: LisBeth is now freely available for download from our website and offers new perspectives of analysis to its users, whether systematicians and biogeographers. We show that the representation of characters offered by LisBeth allows new inferences about the existing relationship between areas of endemism studied. We have highlighted the emergence of a general "pattern" from a joint analysis of 35 unrelated taxa (plants and animals). In a second step we are interested in a major theoretical problem of contemporary biogeography: "*What about the time dimension in historical biogeography?*". How to account unambiguously different time scales during diachronic analyzes? We propose a model based on the 3ia which allows us to consider the temporal dimension and realize, therefore, true analysis of integrative biogeography.

KEY-WORDS : Historical biogeography, integrative biogeography, 3ia, Southeast Asia, West Pacific, pattern, process.