# Molecular Systematics of the Phoxinin GenusPteronotropis (Otophysi: Cypriniformes)

Authors: Richard L. Mayden; Jason S. Allen
Journal: BioMed Research International
 (2015)

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## Abstract

The genusPteronotropis is widely distributed along the gulf slope of eastern North America from Louisiana to Florida and rivers in South Carolina along the Atlantic slope. Pteronotropis have very distinctive, flamboyant coloration. The habitats most frequently associated with these species include heavily vegetated backwater bayous to small sluggish or flowing tannin-stained streams. Although Pteronotropis is recognized as a valid genus, no phylogenetic analysis of all the species has corroborated its monophyly. In recent years, four additional species have been either described or elevated from synonymy: P. merlini, P. grandipinnis, P. stonei, and P. metallicus, with the wide-ranging P. hypselopterus complex. To examine relationships within this genus and test its monophyly, phylogenetic analyses were conducted using two nuclear genes, recombination activating gene 1, RAG1, and the first intron of S7 ribosomal protein gene in both maximum parsimony and Bayesian analyses. In no analysis was Pteronotropis, as currently recognized, recovered as monophyletic without the
inclusion of the currently recognized Notropis harperi, herein referred to as Pteronotropis. Two major clades are supported: one inclusive of P. hubbsi, P. welaka, and P. harperi and the second inclusive of P. signipinnis, P. grandipinnis, P. hypselopterus plus P. merlini sister to P. euryzonus, and P. metallicus plus P. stonei.


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## Body

## 1. Introduction

The subfamily (or family) Leuciscinae includes all cyprinid species in North America, exceptNotemigonus, and species across Eurasia. Many of the species of this North American fauna have been examined in different phylogenetic studies at varying degrees of universality using both morphological and molecular data. Initial morphological studies by Mayden [1] and Coburn and Cavender [2] revealed exciting new relationships and a reclassification of the North American fauna. These studies were followed with several molecular analyses of different major lineages, genera, and species groups that supported many, but not all, of the monophyletic groups previously identified in one or both of the above studies [3–7]. However, not all proposed genera have been examined for species relationships using molecular markers.One such genus in North America with an increasing and intriguing diversity, biology, and geographic distribution, as well as complex taxonomic history, isPteronotropis. This genus contains one of North America’s most colorful shiners.Pteronotropis hubbsi andP. welaka are relatively slender-body species that, in breeding males, possess enlarged dorsal fins, whereas the remaining species,P. euryzonus,P. hypselopterus,P. merlini,P. grandipinnis,P. stonei,P. metallicus, andP. signipinnis, are more deep-bodied and lack enlarged dorsal fins in breeding males. The most frequently associated habitat of these species across their ranges includes deep, backwater bayous, small sluggish tannin-stained streams, and flowing tannin-stained streams, all with ample aquatic vegetation. However, despite several studies on shiners and relatives to date,Pteronotropis has received essentially no recent attention as to their relationships and has been proposed to be an unnatural grouping. Herein, we provide the first examination of phylogenetic relationships of all species in the genus (formerly subgenus ofNotropis [1]) and a test of the monophyly of this purported lineage. Two nuclear genes are used in this analysis because of their previously demonstrated genetic distances and resulting ability to resolve nodes deeper than at the crown of trees. These genes have been used successfully for resolution of more basal lineages of North American cyprinids by several recent papers [3–10].Resulting phylogenetic inferences of species of this group and their eventual placement relative to other North American cyprinids are critical as they largely facilitate more process-level questions as to the evolution of the biology of the species and other lineages to better understand the processes of anagenesis and speciation. While multiple papers listed above have made groundbreaking strides in providing a phylogenetic framework where one previously did not exist for North American cyprinids, Hollingsworth et al. [10] provide an excellent evaluation of a subset of the fauna and a novel hypothesis as to habitat shifts for clades with differing rates of speciation. Given that no study has examined all of the species ofPteronotropis, we provide a review of the history of the genus and molecular phylogenetic analyses of the species using two nuclear genes that result in identical species relationships based on mitochondrial genes in Mayden and Allen [11].Taxonomic History.Pteronotropis currently includes nine species in rivers and streams distributed along the gulf slope from Louisiana to Florida and along the Atlantic slope as far north as South Carolina. One species,P. hubbsi, currently occurs only in southern Arkansas and northern Louisiana but was likely to be more widely distributed in lowland habitats; the conservation status of this species is of concern, as it has not been found in some locations (including southern Illinois) for several decades.In a study focusing on 566 morphological traits of a large number of cyprinids, Mayden [1] elevatedPteronotropis to generic level and includedP. welaka,P. signipinnis,P. hypselopterus, andP. euryzonus within the genus but leftP. hubbsi inNotropis. Recently, Suttkus and Mettee [12], with no characters, phylogenetic analysis, or substantive phylogenetic argument, maintained thatPteronotropis was a subgenus within the genusNotropis (as classified before Mayden’s [1] analysis) and that this subgenus contained onlyP. euryzonus and theP. hypselopterus complex (P. hypselopterus,P. grandipinnis,P. stonei,P. metallicus, andP. merlini).The phylogenetic relationships ofPteronotropis have been somewhat enigmatic over the years. Species share derived and distinctive color patterns that include bright red-orange to yellow striped dorsal, caudal, and anal fins and a broad dark lateral band extending from the head to the caudal peduncle. The genus was divided into two groups based on morphological and molecular characters [1, 4].Pteronotropis signipinnis was described by Bailey and Suttkus [13] and was considered a member of the genusNotropis (subgenusPteronotropis by Fowler [14]), along withP. hypselopterus.Pteronotropis euryzonus [15] was later added to this subgenus and was considered a close relative toP. hypselopterus; however, neither of the above two studies includedP. hubbsi orP. welaka and they were conducted in a prephylogenetic era.Pteronotropis hubbsi was described by Bailey and Robison [16] and was thought to be closely related toP. welaka; at that time, neither species was allocated to the subgenusPteronotropis. In a study utilizing twenty-one allozyme loci, Dimmick [17] examined nine species (mostlyPteronotropis). This allozyme analysis revealedPteronotropis as nonmonophyletic, withP. hubbsi andP. welaka as distantly related andN. signipinnis andN. hypselopterus as sister species. Consequently, Dimmick [17] argued that all of the morphological characters of Bailey and Robison [16], thought to indicate a close relationship betweenP. hubbsi andP. welaka, were the result of convergent evolution.In the first sequence analysis of this group, Simons et al. [18] used mitochondrial cytochromeb gene and failed to corroboratePteronotropis as a monophyletic group. With both parsimony and likelihood analyses,P. euryzonus was sister toP. hypselopterus and an unrelated clade includedP. signipinnis sister toP. hubbsi plusP. welaka. Later, in a subsample ofPteronotropis species, Simons et al. [4], using two mitochondrial genes (12S, 16S), and Bufalino and Mayden [5, 6], using two nuclear loci (RAG1, S7), foundPteronotropis as monophyletic but, again, only with the inclusion of “Notropis”harperi; however, neither of these analyses included all species of the genus. Other early molecular data and analyses also failed to resolve the phylogenetic relationships of the above species that were generally phenetically similar. Most recently a study by Hollingsworth et al. [10], using one mtDNA gene and nDNA genes, corroborated the monophyly of a subsample of species ofPteronotropis that also includedN. harperi.While there have been several efforts testing the monophyly ofPteronotropis, its composition, and at resolving the phylogenetic relationships of species since its elevation to genus, no single study has included all of the species in the genus and appropriate outgroups based on earlier studies and some did not include the morphologically similarNotropis harperi. With the elevation of species from synonymy withP. hypselopterus and the description of a new species [12], the complexity involved in testing the monophyly of the genus and species relationships have become even more biologically interesting. While Suttkus and Mettee [12] did provide dialogue invoking phylogenetic terminology as to species relationships, their study contained no phylogenetic analyses, no discussions of character homology, or any morphological or molecular synapomorphies. To date, no investigation has been completed for this group inclusive of all of the purported species ofPteronotropis. Thus, the objectives of the current study are twofold: (1) testing the monophyly of the genus and (2) examining relationships of all of the purported species of the genus using two nuclear genes.


## 2. Materials and Methods

### 2.1. Specimens and DNA Extraction/Amplification and Alignment

Museum catalogue numbers for vouchers in this study include UAIC (University of Alabama Ichthyological Collection) and SLUM (Saint Louis University Museum). Specimens examined in this study were either frozen at Saint Louis University, preserved in 95% ethanol, or captured alive and transported to Saint Louis University (Table1). Outgroup taxa included species from the generaCyprinella,Lythrurus, andNotropis. Species ofCyprinella were included as outgroup taxa due to previous studies indicating their close relationships toPteronotropis. Because this analysis focuses on nuclear gene variation as it contributes to phylogenetic relationships and the inadequate sampling of all relevant taxa in previous studies, cytochromeb sequences of previous mitochondrial analyses are not included. Genomic DNA was extracted using the QIAGEN QIAamp tissue kit according to the manufacturer’s recommendations (QIAGEN, Valencia, CA). The two nuclear genes included recombination activating gene 1, RAG1, and the first intron of S7 ribosomal protein gene. Both genes were amplified, via PCR, and internal primers amplification and sequencing were developed for S7. These include the forward primers 5′-GCCACTGCAGCCGCCATAAT-3′ and 5′-GCCCCAGCTTTCCACCCATTAC-3′ and reverse primers 5′-CCCGAGGGCTGTGAGGAGTAA-3′ and 5′-CCCCCTCAGCCGCCGACTA-3′. Universal primers for RAG1 and S7 were detailed in López et al. [19] and Chow and Hazama [20], respectively. In addition, both forward and reverse internal primers were developed for S7. For RAG1, each 25 μL PCR reaction consisted of 2 μL of DNTPs, 2.5 μL of 10X Taq buffer, 3 μL of both forward and reverse primers, 10.375 μL of dH2O, 1 μL of Taq polymerase, or .125 μL of HotStartTaq Polymerase (QIAGEN, Valencia, CA). Amplifications consisted of 35 cycles of an initial denaturation of 95°C for 15 minutes with an additional denaturation of 94°C for 40 seconds. This was followed by an annealing temperature of 55°C for 1 minute, an initial extension of 72°C for 90 seconds, and a final extension of 72°C for 5 minutes. Conditions for S7 were identical except the annealing temperature was set at 59°C. For the S7 intron, products that failed to amplify using the universal primers were reamplified using nested PCR reactions with the same conditions except for specific annealing temperatures as specified by the chemistry for the internal primers. Taxa failing to amplify with internal primers were cloned using the pGEM-T Easy Vector System kit (PROMEGA, Madison, WI) as outlined in Lang and Mayden [9]. PCR products were purified using QIAGEN gel extraction kits (QIAGEN, Valencia, CA). Sequencing was performed using a BigDye labeled dideoxy sequencing kit (BigDye) and visualized on an ABI 377 automated sequencer (Auburn University Molecular Genetics Instrumentation Facility, Auburn, AL) or an ABI 3700 (Macrogen Sequencing Facility, Seoul, South Korea). Both the heavy and light strands were sequenced for all samples and the sequences were aligned with Clustal X [21] with reference to the accompanying electropherograms. Some individuals contained heterozygote peaks in the RAG1 data and these heterozygote base pair positions were coded using standard degeneracy codes.Table 1
Species, localities, and GenBank numbers of specimens used for sequencing and analyses of S7 and RAG1.
(a)
Species and drainage
Stream, county, state
Catalogue number
S7
RAG1
Extraction
Pteronotropis euryzonus
Chattahoochee R.
Maringo Cr., Russell, AL
UAIC 12229
KM048270
KJ634252
22
Chattahoochee R.
Snake Cr., Russell, AL
UAIC 10493
KM048276
KJ634258
51
Chattahoochee R.
Snake Cr., Russell, AL
UAIC 10493
KM048277
KJ634259
52
Pteronotropis grandipinnis
Apalachicola R.
Irwin Mill Cr., Houston, AL
No voucher
KM048265
KJ634247
12
Apalachicola R.
Irwin Mill Cr., Houston, AL
No voucher
KM048266
KJ634248
13
Pteronotropis hypselopterus
Mobile R.
Cedar Cr., Mobile, AL
UAIC 12730
KM048256
KJ634238
01
Mobile R.
Cedar Cr., Mobile, AL
UAIC 12730
KM048257
KJ634239
02
Mobile R.
Cedar Cr., Mobile, AL
UAIC 12730
KM048258
KJ634240
03
Alabama R.
Little Reedy Cr., AL
UAIC 14326
KM048269
KJ634251
18
Pteronotropis hubbsi
Ouachita R.
Backwater pond, Ouachita, LA
UAIC 11928
KM048261
KJ634243
06
Ouachita R.
Backwater pond, Ouachita, LA
UAIC 11928
KM048262
KJ634244
07
Little R.
Little R., McCurtain, OK
UAIC 12053
KM048273
KJ634255
41
Pteronotropis merlini
Pea R.
Clearwater Cr., Coffee, AL
No voucher
KM048267
KJ634249
16
Pea R.
Clearwater Cr., Coffee, AL
No voucher
KM048268
KJ634250
17
Pteronotropis metallicus
Suwannee R.
Sampson R., Bradford, FL
UF 158855
KM048278
KJ634260
96
Suwannee R.
Sampson R., Bradford, FL
UF 158855
KM048279
KJ634261
97
Pteronotropis signipinnis
Pascagoula R.
Beaverdam Cr., Forest, MS
UAIC 13416
KM048259
KJ634241
04
Pascagoula R.
Beaverdam Cr., Forest, MS
UAIC 13416
KM048260
KJ634242
05
Mobile R.
Cedar Cr., Mobile, AL
UAIC 12730
KM048271
KJ634253
23
Mobile R.
Cedar Cr., Mobile, AL
UAIC 12730
KM048272
KJ634254
24
Pteronotropis stonei
N. Fork Edisto R.
Murphy Mill Cr., Calhoun, SC
SLUM 1121
KM048281
KJ634263
101
N. Fork Edisto R.
Murphy Mill Cr., Calhoun, SC
SLUM 1121
KM048280
KJ634262
100
Combahee R.
Savannah Cr., Colleton, SC
SLUM 1122
KM048282
KJ634264
102
Pteronotropis welaka
Cahaba R.
Lightsey pond, Bibb, AL
UAIC 10391
KM048263
KJ634245
10
Cahaba R.
Lightsey pond, Bibb, AL
UAIC 10391
KM048264
KJ634246
11
Pearl R.
Lees Cr., Washington, LA
UAIC 12205
KM048274
KJ634256
48
Mobile Bay
Lees Cr., Washington, LA
UAIC 12205
KM048275
KJ634257
49
Pteronotropis harperi
GU134235
GU136332
(b)
Outgroup taxa (note thatPteronotropis harperi was also originally an outgroup species)
Species
S7
RAG1
Cyprinella formosa
GU 134192
GU136293
Lythrurus fumeus
GU134222
GU136231
Lythrurus umbratilis
GU134223
GU136322
Nocomis leptocephalus
GU134236
GU136333
Notropis asperifrons
GU134231
GU136330
Notropis atherinoides
GU134232
EF452832
Notropis blennius
GU134234
GU136331
Notropis leuciodus
GU134237
GU136334
Notropis maculatus
GU134238
GU136335
Notropis
ortenburgeri
GU134240
GU136337
Notropis nazas
GU134239
GU136336
Notropis stilbius
GU134241
GU136338
Notropis volucellus 1
GU134242
GU136339
Notropis volucellus 2
GU134243
GU136340


### 2.2. Phylogenetic Analyses

An incongruence-length difference analysis (ILD [22]) was performed with 1000 replicates to test for incongruence between the RAG1 and S7 data sets. Maximum parsimony (MP) analyses (MPA) were conducted in PAUP
*
4.0
b
10 [23]. All analyses consisted of a heuristic search model with 1000 random addition sequence replicates and TBR. Support for the parsimony analyses was generated using bootstrap analysis (BS) with 1000 bootstrap pseudo-replicates [18]. Bayesian analyses (BA) were conducted in Mr. Bayes 3.0b4 [24]. S7 intron all gaps were treated as missing data. The model of sequence evolution was determined using Modeltest v3.04 [25] with single partitions for each marker; the best-fit model for S7 was HKY + G and that for Rag1 was TrN + I + G. BA included four heated Markov chains using the default temperature setting. Log-likelihood scores were plotted against generation time to establish burn-in; trees prior to stationarity were discarded. Post-burn-in trees were used to develop the 50% majority rule consensus tree. Posterior probabilities (PP) were used as an indication of nodal support in BA.


## 2.1. Specimens and DNA Extraction/Amplification and Alignment

Museum catalogue numbers for vouchers in this study include UAIC (University of Alabama Ichthyological Collection) and SLUM (Saint Louis University Museum). Specimens examined in this study were either frozen at Saint Louis University, preserved in 95% ethanol, or captured alive and transported to Saint Louis University (Table1). Outgroup taxa included species from the generaCyprinella,Lythrurus, andNotropis. Species ofCyprinella were included as outgroup taxa due to previous studies indicating their close relationships toPteronotropis. Because this analysis focuses on nuclear gene variation as it contributes to phylogenetic relationships and the inadequate sampling of all relevant taxa in previous studies, cytochromeb sequences of previous mitochondrial analyses are not included. Genomic DNA was extracted using the QIAGEN QIAamp tissue kit according to the manufacturer’s recommendations (QIAGEN, Valencia, CA). The two nuclear genes included recombination activating gene 1, RAG1, and the first intron of S7 ribosomal protein gene. Both genes were amplified, via PCR, and internal primers amplification and sequencing were developed for S7. These include the forward primers 5′-GCCACTGCAGCCGCCATAAT-3′ and 5′-GCCCCAGCTTTCCACCCATTAC-3′ and reverse primers 5′-CCCGAGGGCTGTGAGGAGTAA-3′ and 5′-CCCCCTCAGCCGCCGACTA-3′. Universal primers for RAG1 and S7 were detailed in López et al. [19] and Chow and Hazama [20], respectively. In addition, both forward and reverse internal primers were developed for S7. For RAG1, each 25 μL PCR reaction consisted of 2 μL of DNTPs, 2.5 μL of 10X Taq buffer, 3 μL of both forward and reverse primers, 10.375 μL of dH2O, 1 μL of Taq polymerase, or .125 μL of HotStartTaq Polymerase (QIAGEN, Valencia, CA). Amplifications consisted of 35 cycles of an initial denaturation of 95°C for 15 minutes with an additional denaturation of 94°C for 40 seconds. This was followed by an annealing temperature of 55°C for 1 minute, an initial extension of 72°C for 90 seconds, and a final extension of 72°C for 5 minutes. Conditions for S7 were identical except the annealing temperature was set at 59°C. For the S7 intron, products that failed to amplify using the universal primers were reamplified using nested PCR reactions with the same conditions except for specific annealing temperatures as specified by the chemistry for the internal primers. Taxa failing to amplify with internal primers were cloned using the pGEM-T Easy Vector System kit (PROMEGA, Madison, WI) as outlined in Lang and Mayden [9]. PCR products were purified using QIAGEN gel extraction kits (QIAGEN, Valencia, CA). Sequencing was performed using a BigDye labeled dideoxy sequencing kit (BigDye) and visualized on an ABI 377 automated sequencer (Auburn University Molecular Genetics Instrumentation Facility, Auburn, AL) or an ABI 3700 (Macrogen Sequencing Facility, Seoul, South Korea). Both the heavy and light strands were sequenced for all samples and the sequences were aligned with Clustal X [21] with reference to the accompanying electropherograms. Some individuals contained heterozygote peaks in the RAG1 data and these heterozygote base pair positions were coded using standard degeneracy codes.Table 1
Species, localities, and GenBank numbers of specimens used for sequencing and analyses of S7 and RAG1.
(a)
Species and drainage
Stream, county, state
Catalogue number
S7
RAG1
Extraction
Pteronotropis euryzonus
Chattahoochee R.
Maringo Cr., Russell, AL
UAIC 12229
KM048270
KJ634252
22
Chattahoochee R.
Snake Cr., Russell, AL
UAIC 10493
KM048276
KJ634258
51
Chattahoochee R.
Snake Cr., Russell, AL
UAIC 10493
KM048277
KJ634259
52
Pteronotropis grandipinnis
Apalachicola R.
Irwin Mill Cr., Houston, AL
No voucher
KM048265
KJ634247
12
Apalachicola R.
Irwin Mill Cr., Houston, AL
No voucher
KM048266
KJ634248
13
Pteronotropis hypselopterus
Mobile R.
Cedar Cr., Mobile, AL
UAIC 12730
KM048256
KJ634238
01
Mobile R.
Cedar Cr., Mobile, AL
UAIC 12730
KM048257
KJ634239
02
Mobile R.
Cedar Cr., Mobile, AL
UAIC 12730
KM048258
KJ634240
03
Alabama R.
Little Reedy Cr., AL
UAIC 14326
KM048269
KJ634251
18
Pteronotropis hubbsi
Ouachita R.
Backwater pond, Ouachita, LA
UAIC 11928
KM048261
KJ634243
06
Ouachita R.
Backwater pond, Ouachita, LA
UAIC 11928
KM048262
KJ634244
07
Little R.
Little R., McCurtain, OK
UAIC 12053
KM048273
KJ634255
41
Pteronotropis merlini
Pea R.
Clearwater Cr., Coffee, AL
No voucher
KM048267
KJ634249
16
Pea R.
Clearwater Cr., Coffee, AL
No voucher
KM048268
KJ634250
17
Pteronotropis metallicus
Suwannee R.
Sampson R., Bradford, FL
UF 158855
KM048278
KJ634260
96
Suwannee R.
Sampson R., Bradford, FL
UF 158855
KM048279
KJ634261
97
Pteronotropis signipinnis
Pascagoula R.
Beaverdam Cr., Forest, MS
UAIC 13416
KM048259
KJ634241
04
Pascagoula R.
Beaverdam Cr., Forest, MS
UAIC 13416
KM048260
KJ634242
05
Mobile R.
Cedar Cr., Mobile, AL
UAIC 12730
KM048271
KJ634253
23
Mobile R.
Cedar Cr., Mobile, AL
UAIC 12730
KM048272
KJ634254
24
Pteronotropis stonei
N. Fork Edisto R.
Murphy Mill Cr., Calhoun, SC
SLUM 1121
KM048281
KJ634263
101
N. Fork Edisto R.
Murphy Mill Cr., Calhoun, SC
SLUM 1121
KM048280
KJ634262
100
Combahee R.
Savannah Cr., Colleton, SC
SLUM 1122
KM048282
KJ634264
102
Pteronotropis welaka
Cahaba R.
Lightsey pond, Bibb, AL
UAIC 10391
KM048263
KJ634245
10
Cahaba R.
Lightsey pond, Bibb, AL
UAIC 10391
KM048264
KJ634246
11
Pearl R.
Lees Cr., Washington, LA
UAIC 12205
KM048274
KJ634256
48
Mobile Bay
Lees Cr., Washington, LA
UAIC 12205
KM048275
KJ634257
49
Pteronotropis harperi
GU134235
GU136332
(b)
Outgroup taxa (note thatPteronotropis harperi was also originally an outgroup species)
Species
S7
RAG1
Cyprinella formosa
GU 134192
GU136293
Lythrurus fumeus
GU134222
GU136231
Lythrurus umbratilis
GU134223
GU136322
Nocomis leptocephalus
GU134236
GU136333
Notropis asperifrons
GU134231
GU136330
Notropis atherinoides
GU134232
EF452832
Notropis blennius
GU134234
GU136331
Notropis leuciodus
GU134237
GU136334
Notropis maculatus
GU134238
GU136335
Notropis
ortenburgeri
GU134240
GU136337
Notropis nazas
GU134239
GU136336
Notropis stilbius
GU134241
GU136338
Notropis volucellus 1
GU134242
GU136339
Notropis volucellus 2
GU134243
GU136340


## 2.2. Phylogenetic Analyses

An incongruence-length difference analysis (ILD [22]) was performed with 1000 replicates to test for incongruence between the RAG1 and S7 data sets. Maximum parsimony (MP) analyses (MPA) were conducted in PAUP
*
4.0
b
10 [23]. All analyses consisted of a heuristic search model with 1000 random addition sequence replicates and TBR. Support for the parsimony analyses was generated using bootstrap analysis (BS) with 1000 bootstrap pseudo-replicates [18]. Bayesian analyses (BA) were conducted in Mr. Bayes 3.0b4 [24]. S7 intron all gaps were treated as missing data. The model of sequence evolution was determined using Modeltest v3.04 [25] with single partitions for each marker; the best-fit model for S7 was HKY + G and that for Rag1 was TrN + I + G. BA included four heated Markov chains using the default temperature setting. Log-likelihood scores were plotted against generation time to establish burn-in; trees prior to stationarity were discarded. Post-burn-in trees were used to develop the 50% majority rule consensus tree. Posterior probabilities (PP) were used as an indication of nodal support in BA.


## 3. Results and Discussion

As the ILD test was nonsignificant for heterogeneity between RAG1 and S7, the gene sequences were analyzed both individually and as a concatenated data set. MP analysis of the aligned 1001 bp of S7 (aligned sequence lengths ranged from 839 to 919 bp) yielded 245 bp parsimony informative sites (12.9%). Analyses of these data resulted in 90 equally parsimonious trees (Figure1; length = 697, CI = 0.803, and RI = 0.875). The more conservative RAG1 sequences included 1521 bp with 151 bp sites (9.9%) being parsimony informative. MP analyses of RAG1 resulted in 46,668 equally parsimonious trees (Figure 1; length = 371 steps, CI = 0.658, and RI = 0.866). Individual BA analyses for each gene resulted in some variations in sister-group relationships but all were consistent and supported the monophyly ofPteronotropis (Figure 2). Both MPA and BA of the combined S7 + RAG1 data recovered identical topologies (Figure 3).Figure 1
Inferred species relationships of species ofPteronotropis based on maximum parsimony analyses of RAG1 (a) and S7 (b). Nodal values indicate bootstrap support.
(a)
(b)Figure 2
Inferred species relationships of speciesPteronotropis based on Bayesian analyses of S7 (a) and Rag1 (b). Nodal values indicate posterior probabilities.
(a)
(b)Figure 3
Inferred species relationships of species ofPteronotropis based on maximum parsimony and Bayesian analyses of combined Rag1 + S7 (a) and Rag1 + S7 (b), respectively. Nodal values indicate posterior probabilities.As in previous studies involving species ofPteronotropis, nuclear sequence variation, neither individual nor combined [5, 6], resolvedPteronotropis as a monophyletic group ifNotropis harperi is excluded from the genus. ConstrainingPteronotropis to be monophyletic in the S7 + RAG1 data set withoutN. harperi resulted in a significantly worse tree (1246 steps). In both BA and MPA,Notropis harperi is resolved as sister toP. welaka within the ingroup, a sister-group relationship with strong PP and BS support (Figures 1 and 2).Pteronotropis hubbsi is resolved as sister to this clade, also with strong PP and BS support. All three of these taxa (P. hubbsi (P. welaka +N. harperi)) are resolved as monophyletic and sister to the remaining species traditionally referred to asPteronotropis (PP 95, bootstrap 75; Figure 2). The strong support for the monophyly of the (P. hubbsi (P. welaka +N. harperi)) clade (Figures 1 and 2) is logical as the three species are phenetically and ecologically similar. They possess aspects of similar body coloration in life when not in breeding condition and have similar habitat associations [5, 26, 27]. They are found in deep pools with ample aquatic vegetation and in areas whereP. welakaandN. harperi are sympatric they are often taken syntopically in a sample (pers. obs.). The authors are unaware of any studies corroborating nest association inN. harperi, as observed inP. welaka andP. hubbsi [28–30]. In light of the relationships presented here and in Bufalino and Mayden [4, 5] and Hollingsworth et al. [10], studies ofN. harperi may reveal ecological and behavioral synapomorphies.In all analyses,P. signipinnis is resolved as sister to a clade of remaining species ofPteronotropis (Figures 1 and 2). In analyses of S7 and S7 + Rag1 data sets, the latter clade formed two clades: one inclusive ofP. hypselopterus,P. grandipinnis, andP. merlini and the other inclusive ofP. euryzonus,P. stonei, andP. metallicus. Resolution of the former clade was not complete in either Rag1 or S7 analyses, but both are fully consistent with the phylogeny recovered with the Rag1 + S7 data set. These relationships are in contrast to those hypothesized by Simons et al. [4] based on 12S and 16S ribosomal RNA sequences whereinP. signipinnis was resolved as sister toP. welaka +P. hubbsi. However, this latter study did not include all of the then or currently known species ofPteronotropis.


## 4. Conclusions

Given the consistent sister-group relationship between formerly recognizedNotropis harperi andPteronotropis welaka, the former species is herein referred to asPteronotropis. Nuclear genes RAG1 and S7 support the long-standing question/hypothesis regarding the monophyly ofPteronotropis and provide new insight into the phylogenetic placement ofPteronotropis harperi and the basal-most relationships between the species groups (P. hubbsi,P. welaka, andP. harperi) relative to the remaining species ofPteronotropis. These relationships are also consistent with those presented by Bailey and Suttkus [13] using mitochondrial gene ND2. In recent years, the general trend in phylogenetics has been to place greater emphasis on the use of nuclear genes, largely because of issues associated with hybridization, intergradation, lineage sorting, and disagreement between gene and species trees [13]. While these nuclear genes have shown a greater ability to resolve relationships at supraspecific levels for this group with greater consistency and stronger branch support, the results presented herein illustrate the benefit in using nuclear genes. However, it is also true that mitochondrial genes have been extremely useful in phylogenetic resolutions [26, 27], and like nuclear genes they also vary in their degree of anagenesis and abilities to resolve trees at different levels of universality. While these and other nuclear genes used in the above-cited papers for Cypriniformes clearly display a reduced phylogenetic signal and are more limited in phylogenetic resolution for relationships of populations and species, they are essential for resolution of deeper nodes. This is to be expected as rates of mutation of many nuclear genes (especially protein coding) are generally not as high as that typically found in most mitochondrial genes.



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