# Species boundaries of Gulf of Mexico vestimentiferans (Polychaeta, Siboglinidae) inferred from mitochondrial genes

Maria Pia Miglietta, Stéphane Hourdez, Dominique A. Cowart, Stephen W.

Schaeffer, Charles Fisher

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► To cite this version:

Maria Pia Miglietta, Stéphane Hourdez, Dominique A. Cowart, Stephen W. Schaeffer, Charles Fisher. Species boundaries of Gulf of Mexico vestimentiferans (Polychaeta, Siboglinidae) inferred from mitochondrial genes. Deep Sea Research Part II: Topical Studies in Oceanography, 2010, 57 (21-23), pp.1916-1925. 10.1016/j.dsr2.2010.05.007. hal-01250940

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# Species boundaries of Gulf of Mexico vestimentiferans (Polychaeta, Siboglinidae) inferred from mitochondrial genes

Maria Pia Miglietta<sup>a,*</sup>, Stephane Hourdez<sup>b</sup>, Dominique A. Cowart<sup>a</sup>, Stephen W. Schaeffer<sup>a</sup>, Charles Fisher<sup>a</sup>

At least six morphospecies of vestimentiferan tubeworms are associated with cold seeps in the Gulf of Mexico (GOM). The physiology and ecology of the two best-studied species from depths above 1000 m in the upper Louisiana slope (*Lamellibrachia luymesi* and *Seepiophila jonesi*) are relatively well understood. The biology of one rare species from the upper slope (escarpiid sp. nov.) and three morphospecies found at greater depths in the GOM (*Lamellibrachia* sp. 1, *L. sp.* 2, and *Escarpia laminata*) are not as well understood. Here we address species distributions and boundaries of cold-seep tubeworms using phylogenetic hypotheses based on two mitochondrial genes. Fragments of the mitochondrial large ribosomal subunit rDNA (16S) and cytochrome oxidase subunit I (COI) genes were sequenced for 167 vestimentiferans collected from the GOM and analyzed in the context of other seep vestimentiferans for which sequence data were available. The analysis supported five monophyletic clades of vestimentiferans in the GOM. Intra-clade variation in both genes was very low, and there was no apparent correlation between the within-clade diversity and collection depth or location. Two of the morphospecies of *Lamellibrachia* from different depths in the GOM could not be distinguished by either mitochondrial gene. Similarly, *E. laminata* could not be distinguished from other described species of *Escarpia* from either the west coast of Africa or the eastern Pacific using COI. We suggest that the mitochondrial COI and 16S genes have little utility as barcoding markers for seep vestimentiferan tubeworms.

## 1. Introduction

For the better part of the last century, marine biologists assumed oceans were largely interconnected by currents that enabled larvae and propagules to reach distant shores and assure gene flow even over great distances. More recently, the use of molecular tools has challenged assumptions regarding population structure and speciation in the ocean and demonstrated that marine animals often have genetically distinct populations despite geographic proximity (Palumbi and Warner, 2003). Although sharp genetic breaks between close populations have been recorded throughout the ocean, most of what is known about speciation patterns and phylogeography has been inferred from shallow-water and coastal systems, which represent only about 15% of the aquatic environment. Thus, our knowledge of processes that lead to population divergence and speciation in the

open ocean is relatively limited (Thornhill et al., 2008, and references therein; Zardus et al., 2006).

Vestimentiferan tubeworms, which include 10 genera in the polychaete family Siboglinidae (Halanych et al., 2001; Kojima et al., 2002; McMullin et al., 2003; Rouse, 2001), are abundant at deep-sea hydrothermal vents and cold seeps at depths ranging from 80 to 9345 m (Cordes et al., 2007b; Mironov, 2000; Miura et al., 2002). In the deep Gulf of Mexico, six morphospecies have been reported (Cordes et al., 2009). Two described species, *Lamellibrachia luymesi* (van der Land and Narrevang, 1975) and *Seepiophila jonesi* (Gardiner et al., 2001), are relatively well studied, and their ecology and physiology are well understood (Bergquist et al., 2002; Cordes et al., 2007a, b). They occur on the upper Louisiana slope at between ~500 and 950 m depth and occasionally co-occur with a rare undescribed species, escarpiid sp. nov. The three other morphological species are found on the lower Louisiana slope at depths greater than about 950 m (*Lamellibrachia* sp. 1, *L. sp.* 2, and *Escarpia laminata*).

In this paper, we present phylogenetic hypotheses based on the mitochondrial large ribosomal subunit rDNA gene (16S) and mitochondrial cytochrome oxidase 1 gene (COI) of over 200

<footnote><sup>a</sup> The Pennsylvania State University, Department of Biology, 208 Mueller Lab, University Park, PA 16802-5301, USA</footnote>

<footnote><sup>b</sup> Station Biologique de Roscoff, CNRS-UPMC, BP74, 29680 Roscoff, France</footnote>

<footnote>* Corresponding author.
E-mail address: mp.miglietta@psu.edu (M. Pia Miglietta).</footnote>
vestimentiferans (sequenced for either or both genes) including 180 individuals from the six morphospecies that occur in the Gulf of Mexico. Phylogenetic trees are used to examine the distribution of vestimentiferans in the Gulf of Mexico and their relations to other vestimentiferans around the world. We examined the concordance between the morphological and phylogenetic data to identify differences between the genealogical and morphological species analyzed. Finally, we compared between- and within-species 16S and COI genetic distances and show that these two mitochondrial genes have little utility as "barcoding molecules" for vestimentiferans.

**2. Material and methods**

**2.1. Collection of material**

Vestimentiferans were collected in the deep Gulf of Mexico from 12 sites on two cruises in 2006 and 2007, using the DSV ALVIN and R.V. *Atlantis* in 2006 and ROV JASON II and the NOAA ship *Ronald Brown* in 2007 (see Fig. 1). Vestimentiferans were collected using either the Bushmaster Jr. collection device (for samples destined also for community ecology analyses, see Cordes et al., 2010) or the submersible manipulators and placed directly into a collection box. Aboadship, all vestimentiferans were identified using morphological criteria, and subsamples of vestimentum tissue were frozen for subsequent analyses at the Pennsylvania State University. Additional frozen vestimentiferan tissue samples collected previously from shallower sites on the upper Louisiana slope using the DSV JOHNSON SEA LINK were also analyzed for this study (see Table 1 for a complete list of specimens).

**2.2. DNA sequencing**

DNA was extracted either by boiling a small amount of frozen tissue in 600 µL of 10% Chelex solution (Bio-Rad) or using a CTAB+PVP method modified from Doyle and Doyle (1987), followed by a standard ethanol precipitation.

A 524 bp fragment of the mitochondrial 16S gene was amplified using primers 16Sar and 16Sbr (Kojima et al., 1995). A 689 bp fragment of the mitochondrial gene COI was amplified using the primers HCO and LCO (Folmer et al., 1994). Amplification was performed under the following PCR conditions: 94 °C (1 min); 50 °C (2 min); and 72 °C (2.5 min) for 30 cycles. All PCR reactions

were performed using 0.5 µl of each primer, 2.5 µl of 10XBuffer, 2 µl of 10 µM dNTPs, 0.2 µl of taq, 16.5 µl of water, and 3 µl of template. The PCR product was first purified with the ExoSap-it protocol (USB, Affimetrix) and then run on a 2% agarose gel stained with ethidium bromide to enable us to check the quantity and quality of the product. The purified PCR product was used as a template for double-stranded sequencing that was carried out at the Pennsylvania State University Sequencing Core Facility, University Park, Pennsylvania, using ABI 3730 sequencer machines.

**2.3. Phylogenetic analysis**

Sequences were first assembled and edited using Geneious Pro 4.0.4 (Biomatters Ltd.), and then aligned using ClustalX (Thompson et al., 2002). All alignments were confirmed and edited visually in MacClade 4.0.6 OS X (Maddison and Maddison, 2000) to insure that indel variation was aligned consistently among all sequenced genes.

Phylogenetic analyses of the aligned sequences were conducted using the maximum parsimony (MP) optimality criterion and neighbor joining (Saitou and Nei, 1987) (NJ) in PAUP* version 4.0b10 for Macintosh (Wilgenbusch and Swofford, 2003), and the maximum likelihood (ML) optimality criterion in GARLI v0.951.OsX-GUI (Zwickl, 2006) and PhyML (Guindon and Gascuel, 2003). The best-fit model used in PhyML and PAUP* was assessed using the akaike information criterion as implemented in modeltest (Posada, 2003; Posada and Crandall, 1998). The best-fit model was (HKY+I+G) for the COI dataset and (GTR+G) for the 16S dataset. Clade stability was assessed by ML bootstrap analysis (Felsenstein, 1985) in GARLI (100 bootstrap replicates) and NJ (1000 replicates) in PAUP*. The ML analyses in GARLI were performed using random starting trees and default termination conditions. Within- and between-species distances were estimated in MEGA 4 (Tamura et al., 2007).

**3. Results**

The complete COI dataset includes 146 sequences (Table 1) of the six Gulf of Mexico (GOM) cold-seep morphospecies, the available GenBank sequences of *E. southwardae*, *E. spicata*, and assorted *Lamellibrachia* species from around the world. Sequences from the hydrothermal vent-dwelling genera *Riftia*, *Oasisia*, *Tevnia*, and *Arcovestia* were used as outgroups. We restricted our analyses to

<figure><img src="image_3.png" /><figcaption>**Fig. 1.** Map of new deep-water collection sites in the Gulf of Mexico.</figcaption></figure>
<table><caption>**Table 1**
Genbank accession numbers and genes analyzed.</caption><thead><tr><th>Sample<sup>a</sup></th><th>Clade</th><th>Location<sup>b</sup></th><th>GenBank Accession #</th><th>Genes</th></tr></thead><tbody><tr><td>1.AC818</td><td>Escarpia laminate</td><td>GOM AC818</td><td>16S: GU068165<br>COI: GU059163</td><td>16S/COI</td></tr><tr><td>2.AC818</td><td>Escarpia laminata</td><td>GOM AC818</td><td>COI: GU059196</td><td>COI</td></tr><tr><td>3.AC818</td><td>Escarpia laminata</td><td>GOM AC818</td><td>16S: GU068166<br>COI: GU059205</td><td>16S/COI</td></tr><tr><td>4.AC818</td><td>Escarpia laminata</td><td>GOM AC818</td><td>16S: GU068167<br>COI: GU059214</td><td>16S/COI</td></tr><tr><td>5.AC818</td><td>Escarpia laminata</td><td>GOM AC818</td><td>16S: GU068168<br>COI: GU059222</td><td>16S/COI</td></tr><tr><td>6.AC818</td><td>Escarpia laminata</td><td>GOM AC818</td><td>16S: GU068169<br>COI: GU059228</td><td>16S/COI</td></tr><tr><td>7.AC818</td><td>Escarpia laminata</td><td>GOM AC818</td><td>16S: GU068170<br>COI: GU059234</td><td>16S/COI</td></tr><tr><td>8.AC818</td><td>Lamellibrachia luymesi/sp. 1</td><td>GOM AC818</td><td>16S: GU068171</td><td>16S</td></tr><tr><td>10.GB697</td><td>Escarpia laminata</td><td>GOM GB697</td><td>16S: GU068172<br>COI: GU059164</td><td>16S/COI</td></tr><tr><td>11.GB829</td><td>Escarpia laminata</td><td>GOM GB829</td><td>16S: GU068173<br>COI: GU059170</td><td>16S/COI</td></tr><tr><td>12.GB829</td><td>Escarpia laminata</td><td>GOM GB829</td><td>16S: GU068174<br>COI: GU059174</td><td>16S/COI</td></tr><tr><td>13.GC600</td><td>Escarpia laminata</td><td>GOM GC600</td><td>16S: GU068175</td><td>16S</td></tr><tr><td>14.GC852</td><td>Escarpia laminata</td><td>GOM GC852</td><td>16S: GU068176<br>COI: GU059185</td><td>16S/COI</td></tr><tr><td>17.GC852</td><td>Escarpia laminata</td><td>GOM GC852</td><td>16S: GU068177<br>COI: GU059192</td><td>16S/COI</td></tr><tr><td>18.GC852</td><td>Escarpia laminata</td><td>GOM GC852</td><td>COI: GU059193</td><td>COI</td></tr><tr><td>19.GC852</td><td>Escarpia laminata</td><td>GOM GC852</td><td>16S: GU068178<br>COI: GU059194</td><td>16S/COI</td></tr><tr><td>19B.AC818</td><td>Escarpia laminata</td><td>GOM AC 818</td><td>COI: GU059195</td><td>COI</td></tr><tr><td>20.WR269</td><td>Escarpia laminata</td><td>GOM WR269</td><td>16S: GU068179<br>COI: GU059197</td><td>16S/COI</td></tr><tr><td>21.WR269</td><td>Escarpia laminata</td><td>GOM WR269</td><td>16S: GU068180<br>COI: GU059198</td><td>16S/COI</td></tr><tr><td>22.WR269</td><td>Escarpia laminata</td><td>GOM WR269</td><td>16S: GU068181<br>COI: GU059199</td><td>16S/COI</td></tr><tr><td>23.WR269</td><td>Escarpia laminata</td><td>GOM WR269</td><td>COI: GU059200</td><td>COI</td></tr><tr><td>24.WR269</td><td>Escarpia laminata</td><td>GOM WR269</td><td>COI: GU059201</td><td>COI</td></tr><tr><td>26.AT340</td><td>Escarpia laminata</td><td>GOM AT340</td><td>16S: GU068182</td><td>16S</td></tr><tr><td>27.AT340</td><td>Escarpia laminata</td><td>GOM AT340</td><td>16S: GU068183<br>COI: GU059202</td><td>16S/COI</td></tr><tr><td>28.AT340</td><td>Escarpia laminata</td><td>GOM AT340</td><td>16S: GU068184<br>COI: GU059203</td><td>16S/COI</td></tr><tr><td>29.AT340</td><td>Escarpia laminata</td><td>GOM AT340</td><td>16S: GU068185<br>COI: GU059204</td><td>16S/COI</td></tr><tr><td>30.AT340</td><td>Escarpia laminata</td><td>GOM AT340</td><td>16S: GU068186<br>COI: GU059206</td><td>16S/COI</td></tr><tr><td>31.AT340</td><td>Escarpia laminata</td><td>GOM AT340</td><td>16S: GU068187<br>COI: GU059207</td><td>16S/COI</td></tr><tr><td>32.AT340</td><td>Escarpia laminata</td><td>GOM AT340</td><td>16S: GU068188<br>COI: GU059208</td><td>16S/COI</td></tr><tr><td>33.AT340</td><td>Escarpia laminata</td><td>GOM AT340</td><td>16S: GU068189<br>COI: GU059209</td><td>16S/COI</td></tr><tr><td>34.WR264</td><td>Escarpia laminata</td><td>GOM WR269</td><td>16S: GU068190</td><td>16S</td></tr><tr><td>35.WR269</td><td>Escarpia laminata</td><td>GOM WR269</td><td>16S: GU068191<br>COI: GU059210</td><td>16S/COI</td></tr><tr><td>37.AC601</td><td>Escarpia laminata</td><td>GOM AC601</td><td>16S: GU068192<br>COI: GU059211</td><td>16S/COI</td></tr><tr><td>38.AC601</td><td>Escarpia laminata</td><td>GOM AC601</td><td>16S: GU068193<br>COI: GU059212</td><td>16S/COI</td></tr><tr><td>39.AC601</td><td>Escarpia laminata</td><td>GOM AC601</td><td>16S: GU068194<br>COI: GU059213</td><td>16S/COI</td></tr><tr><td>40.AC601</td><td>Escarpia laminata</td><td>GOM AC601</td><td>16S: GU068195<br>COI: GU059215</td><td>16S/COI</td></tr><tr><td>41.AC601</td><td>Escarpia laminata</td><td>GOM AC601</td><td>16S: GU068196</td><td>16S</td></tr><tr><td>42.AC601</td><td>Escarpia laminata</td><td>GOM AC602</td><td>16S: GU068197<br>COI: GU059216</td><td>16S/COI</td></tr><tr><td>43.AC601</td><td>Escarpia laminata</td><td>GOM AC601</td><td>16S: GU068198<br>COI: GU059217</td><td>16S/COI</td></tr><tr><td>44.AC601</td><td>Escarpia laminata</td><td>GOM AC602</td><td>16S: GU068199<br>COI: GU059218</td><td>16S/COI</td></tr><tr><td>45.AC601</td><td>Escarpia laminata</td><td>GOM AC601</td><td>16S: GU068200<br>COI: GU059219</td><td>16S/COI</td></tr><tr><td>46.AT340</td><td>Escarpia laminata</td><td>GOM AT340</td><td>16S: GU068201</td><td>16S</td></tr><tr><td>47.AC601</td><td>Escarpia laminata</td><td>GOM AC601</td><td>16S: GU068202<br>COI: GU059220</td><td>16S/COI</td></tr></tbody></table>
<table><caption>Table 1 (continued)</caption><thead><tr><th>Sample<sup>a</sup></th><th>Clade</th><th>Location<sup>b</sup></th><th>GenBank Accession #</th><th>Genes</th></tr></thead><tbody><tr><td>48.AC601</td><td>Escarpia laminata</td><td>GOM AC601</td><td>16S: GU068203</td><td>16S</td></tr><tr><td>49.AC601</td><td>Escarpia laminata</td><td>GOM AC601</td><td>16S: GU068204<br>COI: GU059221</td><td>16S/COI</td></tr><tr><td>50.AC601</td><td>Escarpia laminata</td><td>GOM AC601</td><td>16S: GU068205<br>COI: GU059223</td><td>16S/COI</td></tr><tr><td>51.AT340</td><td>Escarpia laminata</td><td>GOM AT340</td><td>16S: GU068206</td><td>16S</td></tr><tr><td>52.AT340</td><td>Escarpia laminata</td><td>GOM AT340</td><td>16S: GU068207</td><td>16S</td></tr><tr><td>54.AC601</td><td>Escarpia laminata</td><td>GOM AC601</td><td>16S: GU068208<br>COI: GU059224</td><td>16S/COI</td></tr><tr><td>55.L. luymesi BH</td><td>Lamellibrachia luymesi/sp. 1</td><td>GOM GC185</td><td>16S: GU068209<br>COI: GU059225</td><td>16S/COI</td></tr><tr><td>56.L. sp. 1 GB697</td><td>Lamellibrachia luymesi/sp. 1</td><td>GOM GB697</td><td>16S: GU068210</td><td>16S</td></tr><tr><td>57.L. luymesi GC234</td><td>Lamellibrachia luymesi/sp. 1</td><td>GOM GC234</td><td>16S: GU068211<br>COI: GU059226</td><td>16S/COI</td></tr><tr><td>58.L. sp. 1 GC852</td><td>Lamellibrachia luymesi/sp. 1</td><td>GOM GC852</td><td>16S: GU068212<br>COI: GU059227</td><td>16S/COI</td></tr><tr><td>59.L. sp. 1 AC601</td><td>Lamellibrachia luymesi/sp. 1</td><td>GOM AC601</td><td>16S: GU068213</td><td>16S</td></tr><tr><td>60.L. luymesi BH</td><td>Lamellibrachia luymesi/sp. 1</td><td>GOM GC185</td><td>16S: GU068214</td><td>16S</td></tr><tr><td>61.L. luymesi BH</td><td>Lamellibrachia luymesi/sp. 1</td><td>GOM GC185</td><td>16S: GU068215</td><td>16S</td></tr><tr><td>62.L. luymesi BH</td><td>Lamellibrachia luymesi/sp. 1</td><td>GOM GC185</td><td>16S: GU068216<br>COI: GU059229</td><td>16S/COI</td></tr><tr><td>63.L. luymesi BH</td><td>Lamellibrachia luymesi/sp. 1</td><td>GOM GC185</td><td>16S: GU068217</td><td>16S</td></tr><tr><td>64.L. luymesi BH</td><td>Lamellibrachia luymesi/sp. 1</td><td>GOM GC185</td><td>16S: GU068218<br>COI: GU059230</td><td>16S/COI</td></tr><tr><td>65.L. luymesi BH</td><td>Lamellibrachia luymesi/sp. 1</td><td>GOM GC185</td><td>16S: GU068219</td><td>16S</td></tr><tr><td>66.L. luymesi BH</td><td>Lamellibrachia luymesi/sp. 1</td><td>GOM GC185</td><td>16S: GU068220<br>COI: GU059231</td><td>16S/COI</td></tr><tr><td>67.L. luymesi BH</td><td>Lamellibrachia luymesi/sp. 1</td><td>GOM GC185</td><td>16S: GU068221</td><td>16S</td></tr><tr><td>68.L. luymesi BH</td><td>Lamellibrachia luymesi/sp. 1</td><td>GOM GC185</td><td>16S: GU068222<br>COI: GU059232</td><td>16S/COI</td></tr><tr><td>69.L. luymesi BH</td><td>Lamellibrachia luymesi/sp. 1</td><td>GOM GC185</td><td>16S: GU068223<br>COI: GU059233</td><td>16S/COI</td></tr><tr><td>70.L. luymesi BH</td><td>Lamellibrachia luymesi/sp. 1</td><td>GOM GC185</td><td>16S: GU068224<br>COI: GU059235</td><td>16S/COI</td></tr><tr><td>71.L. luymesi BH</td><td>Lamellibrachia luymesi/sp. 1</td><td>GOM GC185</td><td>16S: GU068225</td><td>16S</td></tr><tr><td>72.L. luymesi BP</td><td>Lamellibrachia luymesi/sp. 1</td><td>GOM GC233</td><td>16S: GU068226<br>COI: GU059236</td><td>16S/COI</td></tr><tr><td>73.L. sp. 1 GB697</td><td>Lamellibrachia luymesi/sp. 1</td><td>GOM GB697</td><td>16S: GU068227<br>COI: GU059237</td><td>16S/COI</td></tr><tr><td>74.L. sp. 1 GB697</td><td>Lamellibrachia luymesi/sp. 1</td><td>GOM GB697</td><td>16S: GU068228</td><td>16S</td></tr><tr><td>75.L. sp. 1 GB697</td><td>Lamellibrachia luymesi/sp. 1</td><td>GOM GB697</td><td>16S: GU068229</td><td>16S</td></tr><tr><td>76.L. sp. 1 GB829</td><td>Lamellibrachia luymesi/sp. 1</td><td>GOM GB829</td><td>16S: GU068230<br>COI: GU059238</td><td>16S/COI</td></tr><tr><td>77.L. sp. 1 GB829</td><td>Lamellibrachia luymesi/sp. 1</td><td>GOM GB829</td><td>16S: GU068231</td><td>16S</td></tr><tr><td>78.L. sp. 1 GB829</td><td>Lamellibrachia luymesi/sp. 1</td><td>GOM GB829</td><td>16S: GU068232<br>COI: GU059239</td><td>16S</td></tr><tr><td>79.L. sp. 1 GB829</td><td>Lamellibrachia luymesi/sp. 1</td><td>GOM GB829</td><td>16S: GU068233</td><td>16S</td></tr><tr><td>80.L. sp. 1 GB829</td><td>Lamellibrachia luymesi/sp. 1</td><td>GOM GB829</td><td>16S: GU068234</td><td>16S</td></tr><tr><td>81.L. luymesi GC234</td><td>Lamellibrachia luymesi/sp. 1</td><td>GOM GC234</td><td>16S: GU068235</td><td>16S</td></tr><tr><td>83.L. luymesi GC234</td><td>Lamellibrachia luymesi/sp. 1</td><td>GOM GC234</td><td>16S: GU068236<br>COI: GU059240</td><td>16S/COI</td></tr><tr><td>84.L. luymesi GC234</td><td>Lamellibrachia luymesi/sp. 1</td><td>GOM GC234</td><td>16S: GU068237</td><td>16S</td></tr><tr><td>85.L. luymesi GC234</td><td>Lamellibrachia luymesi/sp. 1</td><td>GOM GC234</td><td>16S: GU068238</td><td>16S</td></tr><tr><td>86.L. luymesi GC234</td><td>Lamellibrachia luymesi/sp. 1</td><td>GOM GC234</td><td>16S: GU068239<br>COI: GU059241</td><td>16S/COI</td></tr><tr><td>88.L. sp. 1 GC600</td><td>Lamellibrachia luymesi/sp. 1</td><td>GOM GC600</td><td>16S: GU068240<br>COI: GU059242</td><td>16S/COI</td></tr><tr><td>89.L. sp. 1 GC600</td><td>Lamellibrachia luymesi/sp. 1</td><td>GOM GC600</td><td>16S: GU068241<br>COI: GU059243</td><td>16S/COI</td></tr><tr><td>90.L. sp. 1 GC852</td><td>Lamellibrachia luymesi/sp. 1</td><td>GOM GC852</td><td>16S: GU068242<br>COI: GU059244</td><td>16S/COI</td></tr><tr><td>91.L. sp. 1 GC852</td><td>Lamellibrachia luymesi/sp. 1</td><td>GOM GC852</td><td>16S: GU068243</td><td>16S</td></tr><tr><td>92.L. sp. 1 GC852</td><td>Lamellibrachia luymesi/sp. 1</td><td>GOM GC852</td><td>16S: GU068244<br>COI: GU059245</td><td>16S</td></tr><tr><td>93.L. luymesi BH</td><td>Lamellibrachia luymesi/sp. 1</td><td>GOM GC185</td><td>16S: GU068245<br>COI: GU059246</td><td>16S/COI</td></tr><tr><td>94.L. luymesi BH</td><td>Lamellibrachia luymesi/sp. 1</td><td>GOM GC185</td><td>16S: GU068246</td><td>16S</td></tr><tr><td>95.L. luymesi BH</td><td>Lamellibrachia luymesi/sp. 1</td><td>GOM GC185</td><td>16S: GU068247</td><td>16S</td></tr><tr><td>96.L. luymesi BH</td><td>Lamellibrachia luymesi/sp. 1</td><td>GOM GC185</td><td>16S: GU068248</td><td>16S</td></tr><tr><td>97.L. luymesi GC234</td><td>Lamellibrachia luymesi/sp. 1</td><td>GOM GC234</td><td>16S: GU068249</td><td>16S</td></tr><tr><td>98.L. luymesi GC234</td><td>Lamellibrachia luymesi/sp. 1</td><td>GOM GC234</td><td>16S: GU068250</td><td>16S</td></tr><tr><td>99.L. luymesi GC234</td><td>Lamellibrachia luymesi/sp. 1</td><td>GOM GC234</td><td>16S: GU068251<br>COI: GU059247</td><td>16S/COI</td></tr><tr><td>100.L. sp. 1. WR269</td><td>Lamellibrachia luymesi/sp. 1</td><td>GOM WR269</td><td>16S: GU068252</td><td>16S</td></tr><tr><td>102.L. sp. 1 WR269</td><td>Lamellibrachia luymesi/sp. 1</td><td>GOM WR269</td><td>16S: GU068253<br>COI: GU059165</td><td>16S/COI</td></tr></tbody></table>
<table><caption>Table 1 (continued)</caption><thead><tr><th>Sample<sup>a</sup></th><th>Clade</th><th>Location<sup>b</sup></th><th>GenBank Accession #</th><th>Genes</th></tr></thead><tbody><tr><td>103.L. sp. 1 AT340</td><td>Lamellibrachia luymesi/sp. 1</td><td>GOM AT340</td><td>16S: GU068254<br>COI: GU059166</td><td>16S/COI</td></tr><tr><td>104.L. sp. 1 WR269</td><td>Lamellibrachia luymesi/sp. 1</td><td>GOM WR269</td><td>16S: GU068255<br>COI: GU059167</td><td>16S/COI</td></tr><tr><td>105.L. sp. 1 WR269</td><td>Lamellibrachia luymesi/sp. 1</td><td>GOM WR269</td><td>16S: GU068256<br>COI: GU059168</td><td>16S/COI</td></tr><tr><td>107.L. sp. 1 AC601</td><td>Lamellibrachia luymesi/sp. 1</td><td>GOM AC601</td><td>16S: GU068257<br>COI: GU059169</td><td>16S/COI</td></tr><tr><td>110.L. sp. 1 AC601</td><td>Lamellibrachia luymesi/sp. 1</td><td>GOM AC601</td><td>16S: GU068258<br>COI: GU059171</td><td>16S/COI</td></tr><tr><td>112.GB697</td><td>Lamellibrachia sp. 2</td><td>GOM GB697</td><td>16S: GU068259</td><td>16S</td></tr><tr><td>113.GB697</td><td>Lamellibrachia sp. 2</td><td>GOM GB697</td><td>16S: GU068260<br>COI: GU059172</td><td>16S/COI</td></tr><tr><td>114.GB697</td><td>Lamellibrachia sp. 2</td><td>GOM GB297</td><td>16S: GU068261</td><td>16S</td></tr><tr><td>115.GB697</td><td>Lamellibrachia sp. 2</td><td>GOM GB297</td><td>16S: GU068262</td><td>16S</td></tr><tr><td>116.GB829</td><td>Lamellibrachia sp. 2</td><td>GOM GB829</td><td>16S: GU068263</td><td>16S</td></tr><tr><td>117.GC600</td><td>Lamellibrachia sp. 2</td><td>GOM GC600</td><td>16S: GU068264</td><td>16S</td></tr><tr><td>118.GC852</td><td>Lamellibrachia sp. 2</td><td>GOM GC852</td><td>16S: GU068265<br>COI: GU059173</td><td>16S/COI</td></tr><tr><td>119.GC852</td><td>Lamellibrachia sp. 2</td><td>GOM GC852</td><td>16S: GU068266</td><td>16S</td></tr><tr><td>120.GC852</td><td>Lamellibrachia sp. 2</td><td>GOM GC852</td><td>16S: GU068267</td><td>16S</td></tr><tr><td>121.WR269</td><td>Lamellibrachia sp. 2</td><td>GOM WR269</td><td>16S: GU068268</td><td>16S</td></tr><tr><td>122.AT340</td><td>Lamellibrachia sp. 2</td><td>GOM AT340</td><td>16S: GU068269<br>COI: GU059175</td><td>16S/COI</td></tr><tr><td>123.WR2695</td><td>Lamellibrachia sp. 2</td><td>GOM WR269</td><td>16S: GU068270<br>COI: GU059176</td><td>16S/COI</td></tr><tr><td>124.AC601</td><td>Lamellibrachia sp. 2</td><td>GOM AC601</td><td>16S: GU059177</td><td>COI</td></tr><tr><td>126.AC601</td><td>Lamellibrachia sp. 2</td><td>GOM AC601</td><td>16S: GU059178</td><td>COI</td></tr><tr><td>128.L. sp. 1 AT340</td><td>Lamellibrachia luymesi/sp. 1</td><td>GOM AT340</td><td>16S: GU068271<br>COI: GU059179</td><td>16S/COI</td></tr><tr><td>130.GB697</td><td>Seepiophila jonesi</td><td>GOM GB697</td><td>16S: GU068272<br>COI: GU059180</td><td>16S/COI</td></tr><tr><td>131.GB647</td><td>Seepiophila jonesi</td><td>GOM GB647</td><td>16S: GU068273<br>COI: GU05981</td><td>16S/COI</td></tr><tr><td>132.GC234</td><td>Seepiophila jonesi</td><td>GOM GC234</td><td>16S: GU068274<br>COI: GU059182</td><td>16S/COI</td></tr><tr><td>133.GC234</td><td>Seepiophila jonesi</td><td>GOM GC234</td><td>16S: GU068275</td><td>16S</td></tr><tr><td>134.GC234</td><td>Seepiophila jonesi</td><td>GOM GC234</td><td>16S: GU068276<br>COI: GU059183</td><td>16S/COI</td></tr><tr><td>134b.GC234</td><td>Seepiophila jonesi</td><td>GOM GC234</td><td>16S: GU068277<br>COI: GU059184</td><td>16S/COI</td></tr><tr><td>135.GC234</td><td>Seepiophila jonesi</td><td>GOM GC234</td><td>16S: GU068278</td><td>16S</td></tr><tr><td>136.GC234</td><td>Seepiophila jonesi</td><td>GOM GC234</td><td>16S: GU068279</td><td>16S</td></tr><tr><td>137.BH</td><td>Seepiophila jonesi</td><td>GOM GC185</td><td>16S: GU068280</td><td>16S</td></tr><tr><td>138.BH</td><td>Seepiophila jonesi</td><td>GOM GC185</td><td>16S: GU068281</td><td>16S</td></tr><tr><td>139.GC234</td><td>Seepiophila jonesi</td><td>GOM GC234</td><td>16S: GU068282</td><td>16S</td></tr><tr><td>140. GC234</td><td>Seepiophila jonesi</td><td>GOM GC234</td><td>16S: GU068283</td><td>16S</td></tr><tr><td>141.GB647</td><td>Seepiophila jonesi</td><td>GOM GB647</td><td>16S: GU068284<br>COI: GU059186</td><td>16S/COI</td></tr><tr><td>142.GB647</td><td>Seepiophila jonesi</td><td>GOM GB647</td><td>16S: GU068285</td><td>16S</td></tr><tr><td>143.GB647</td><td>Seepiophila jonesi</td><td>GOM GB647</td><td>16S: GU068286<br>COI: GU059187</td><td>16S/COI</td></tr><tr><td>144.GB647</td><td>Seepiophila jonesi</td><td>GOM GB647</td><td>16S: GU068287<br>COI: GU059188</td><td>16S/COI</td></tr><tr><td>145.AC818</td><td>Escarpia laminata</td><td>GOM AC818</td><td>16S: GU068288<br>COI: GU059189</td><td>16S/COI</td></tr><tr><td>146.BH</td><td>Seepiophila jonesi</td><td>GOM GC185</td><td>16S: GU068289</td><td>16S</td></tr><tr><td>147.GB647</td><td>Seepiophila jonesi</td><td>GOM GB647</td><td>16S: GU059190</td><td>COI</td></tr><tr><td>148.GB647</td><td>Seepiophila jonesi</td><td>GOM GB647</td><td>16S: GU059191</td><td>COI</td></tr><tr><td>149.AC601</td><td>Escarpia laminata</td><td>GOM AC601</td><td>16S: GU059248</td><td>COI</td></tr><tr><td>151.GB697</td><td>Lamellibrachia luymesi</td><td>GOM GB697</td><td>16S: GU059250</td><td>COI</td></tr><tr><td>152.GC234</td><td>Lamellibrachia luymesi/sp. 1</td><td>GOM GC234</td><td>16S: GU059253</td><td>COI</td></tr><tr><td>153.GC600</td><td>Lamellibrachia luymesi/sp. 1</td><td>GOM GC600</td><td>16S: GU059254</td><td>COI</td></tr><tr><td>154.NewEscarpidGB485</td><td>Escarpid sp. nov.</td><td>GOM GB425</td><td>16S: GU068290<br>COI: GU059255</td><td>16S/COI</td></tr><tr><td>155.NewEscarpidGC234</td><td>Escarpid sp. nov.</td><td>GOM GC234</td><td>16S: GU068291<br>COI: GU059256</td><td>16S/COI</td></tr><tr><td>157.L. sp. 1 GB697</td><td>Lamellibrachia luymesi/sp. 1</td><td>GOM GB697</td><td>16S: GU059229</td><td>COI</td></tr><tr><td>159.GB697</td><td>Seepiophila jonesi</td><td>GOM GB697</td><td>16S: GU059251</td><td>COI</td></tr><tr><td>160.GB697</td><td>Seepiophila jonesi</td><td>GOM GB697</td><td>16S: GU059252</td><td>COI</td></tr><tr><td>161.GC234</td><td>Lamellibrachia luymesi/sp. 1</td><td>GOM GC234</td><td>xxx-xxx</td><td>COI</td></tr><tr><td>162.GC600</td><td>Lamellibrachia luymesi/sp. 1</td><td>GOM GC600</td><td>xxx-xxx</td><td>COI</td></tr><tr><td>165.GC852</td><td>Lamellibrachia sp. 2</td><td>GOM GC852</td><td>xxx-xxx</td><td>COI</td></tr><tr><td>166.L.sp1 AT340</td><td>Lamellibrachia luymesi/sp. 1</td><td>GOM AT340</td><td>xxx-xxx</td><td>COI</td></tr><tr><td>S. jonesi BH</td><td>Seepiophila jonesi</td><td>GOM GC185</td><td>AF317287</td><td>COI</td></tr><tr><td>S. jonesi GB425</td><td>Seepiophila jonesi</td><td>GOM GB425</td><td>AF317288</td><td>COI</td></tr></tbody></table>
<table><caption>Table 1 (continued)</caption><thead><tr><th>Sample<sup>a</sup></th><th>Clade</th><th>Location<sup>b</sup></th><th>GenBank Accession #</th><th>Genes</th></tr></thead><tbody><tr><td>Lamluymesi GC234</td><td>Lamellibrachia luymesi</td><td>GOM GC234</td><td>AY129136</td><td>COI</td></tr><tr><td>Basibranchia mariana 1</td><td>Basibranchia mariana</td><td>West Pacific</td><td>U74078</td><td>COI</td></tr><tr><td>Arcovestia</td><td>Arcovestia ivanovi</td><td>West Pacific</td><td>AB073491</td><td>COI</td></tr><tr><td>E. laminata</td><td>Escarpia laminata</td><td>West Atlantic</td><td>U74063</td><td>COI</td></tr><tr><td>E. southwardae 1</td><td>Escarpia southwardae</td><td>West Africa</td><td>AY326304</td><td>COI</td></tr><tr><td>E. southwardae 2</td><td>Escarpia southwardae</td><td>West Africa</td><td>AY326303</td><td>COI</td></tr><tr><td>E. spicata</td><td>Escarpia spicata</td><td>East Pacific</td><td>U84262</td><td>COI</td></tr><tr><td>L. sp.1_b</td><td>Lamellibrachia luymesi/sp. 1</td><td>GOM AT340</td><td>U74061</td><td>COI</td></tr><tr><td>OasisiaHaploA</td><td>Oasisia alvinae</td><td>East Pacific</td><td>AY646001</td><td>COI</td></tr><tr><td>OasisiaHaploP</td><td>Oasisia alvinae</td><td>East Pacific</td><td>AY646016</td><td>COI</td></tr><tr><td>Lam.2000Nanaki</td><td>Lamellibrachia sp.</td><td>West Pacific</td><td>D50592</td><td>COI</td></tr><tr><td>Lam.300Sagami</td><td>Lamellibrachia sp.</td><td>West Pacific</td><td>AB088674</td><td>COI</td></tr><tr><td>Lam.300Sagami 1</td><td>Lamellibrachia sp.</td><td>West Pacific</td><td>D38029</td><td>COI</td></tr><tr><td>Lam.barhami10b</td><td>Lamellibrachia barhami</td><td>East Pacific</td><td>AY129137</td><td>COI</td></tr><tr><td>Lam.barhami11b</td><td>Lamellibrachia barhami</td><td>East Pacific</td><td>AY129138</td><td>COI</td></tr><tr><td>Lam.barhami4b</td><td>Lamellibrachia barhami</td><td>East Pacific</td><td>AY129147</td><td>COI</td></tr><tr><td>Lam.barhami7</td><td>Lamellibrachia barhami</td><td>East Pacific</td><td>AY129146</td><td>COI</td></tr><tr><td>Lam.barhami8b</td><td>Lamellibrachia barhami</td><td>East Pacific</td><td>AY129145</td><td>COI</td></tr><tr><td>Lam.barhami9</td><td>Lamellibrachia barhami</td><td>East Pacific</td><td>AY129141</td><td>COI</td></tr><tr><td>Lam.barhamib</td><td>Lamellibrachia barhami</td><td>East Pacific</td><td>U74054</td><td>COI</td></tr><tr><td>L. barhami2</td><td>Lamellibrachia barhami</td><td>East Pacific</td><td>AF315045</td><td>16S</td></tr><tr><td>L. barhami3</td><td>Lamellibrachia barhami</td><td>East Pacific</td><td>AF315045</td><td>16S</td></tr><tr><td>Lam.columna</td><td>Lamellibrachia columna</td><td>West Pacific</td><td>U74061</td><td>COI</td></tr><tr><td>Lam.columna 1</td><td>Lamellibrachia columna</td><td>West Pacific</td><td>AB055210</td><td>COI</td></tr><tr><td>Lam.juni</td><td>Lamellibrachia juni</td><td>West Pacific</td><td>AB242858</td><td>COI</td></tr><tr><td>Lam.juniHaplo1</td><td>Lamellibrachia juni</td><td>West Pacific</td><td>AB264601</td><td>COI</td></tr><tr><td>Lam.juniHaplo2</td><td>Lamellibrachia juni</td><td>West Pacific</td><td>AB264602</td><td>COI</td></tr><tr><td>Lam.juniHaplo3</td><td>Lamellibrachia juni</td><td>West Pacific</td><td>AB264603</td><td>COI</td></tr><tr><td>Lam.juniHaplo4</td><td>Lamellibrachia juni</td><td>West Pacific</td><td>AB264604</td><td>COI</td></tr><tr><td>Lam.juniHaplo5</td><td>Lamellibrachia juni</td><td>West Pacific</td><td>AB264605</td><td>COI</td></tr><tr><td>LamL4</td><td>Lamellibrachia sp.</td><td>West Pacific</td><td>AB055209</td><td>COI</td></tr><tr><td>LamL5</td><td>Lamellibrachia sp.</td><td>West Pacific</td><td>AB055210</td><td>COI</td></tr><tr><td>LamL6</td><td>Lamellibrachia sp.</td><td>West Pacific</td><td>AB088674</td><td>COI</td></tr><tr><td>LamL7</td><td>Lamellibrachia sp.</td><td>West Pacific</td><td>AB088675</td><td>COI</td></tr><tr><td>LamluymesiBH 2</td><td>Lamellibrachia luymesi</td><td>GOM GC185</td><td>AY129133</td><td>COI</td></tr><tr><td>Lam.luymesiBHb</td><td>Lamellibrachia luymesi</td><td>GOM GC185</td><td>AY129132</td><td>COI</td></tr><tr><td>Lam.luymesiBP</td><td>Lamellibrachia luymesi</td><td>GOM GC233</td><td>AY129139</td><td>COI</td></tr><tr><td>Lam.luymesiGB4252</td><td>Lamellibrachia luymesi</td><td>GOM GB425</td><td>AY129135</td><td>COI</td></tr><tr><td>Lam.luymesiGC354</td><td>Lamellibrachia luymesi</td><td>GOM GC354</td><td>AY129126</td><td>COI</td></tr><tr><td>Lam.luymesi VK</td><td>Lamellibrachia luymesi</td><td>GOM VK826</td><td>AY129124</td><td>COI</td></tr><tr><td>Lam.Med</td><td>Lamellibrachia sp. from Med.</td><td>Mediterranean</td><td>EU046616</td><td>COI</td></tr><tr><td>Lam.satsumab</td><td>Lamellibrachia satsuma</td><td>West Pacific</td><td>AF342671</td><td>COI</td></tr><tr><td>NewEscarpiidGB425</td><td>Escarpiid sp. nov.</td><td>GOM GB425</td><td>AY129134</td><td>COI</td></tr><tr><td>Oasisia fujikurai</td><td>Oasisia fujikurai</td><td>South/West Pacific</td><td>AB242857</td><td>COI</td></tr><tr><td>Paraescarpia</td><td>Paraescarpia cf. echinospica</td><td>West Pacific</td><td>D50594</td><td>COI</td></tr><tr><td>Ridgeia</td><td>Ridgeia piscesae</td><td>Juan de Fuca Ridge</td><td>AF022233</td><td>COI</td></tr><tr><td>Ridgeia</td><td>Ridgeia piscesae</td><td>Juan de Fuca Ridge</td><td>AF315054</td><td>16S</td></tr><tr><td>Ridgeia 2</td><td>Ridgeia piscesae</td><td>Juan de Fuca Ridge</td><td>AF315051</td><td>16S</td></tr><tr><td>Ridgeia3</td><td>Ridgeia piscesae</td><td>Juan de Fuca Ridge</td><td>AF315054</td><td>16S</td></tr><tr><td>Riftia</td><td>Riftia pachyptila</td><td>East Pacific</td><td>AY645989</td><td>COI</td></tr><tr><td>Tevnia jerichonana</td><td>Tevnia jerichonana</td><td>East Pacific</td><td>16S: AF315042<br>COI: AY645995</td><td>16S/COI</td></tr><tr><td>S. jonesi BH</td><td>Seepiophila jonesi</td><td>GOM GC185</td><td>AF317287</td><td>COI</td></tr><tr><td>S. jonesi GB425</td><td>Seepiophila jonesi</td><td>GOM GB425</td><td>AF317288</td><td>COI</td></tr></tbody></table>

the species' boundaries for *Lamellibrachia*, *Escarpia*, and *Seepiophila*, and we do not infer higher level phylogenetic relationships among genera because neither 16S nor COI offers sufficient resolution at deeper nodes. The complete and aligned COI dataset included 690 bp, of which 460 were invariant sites, 207 were phylogenetically informative sites, and 23 were autapomorphies.

The complete 16S dataset consisted of 133 sequences (see Table 1 for the complete list of samples), 127 of which were from the Gulf of Mexico. Sequences from the vent-dwelling genera *Tevnia* and *Ridgeia* were used as outgroups. The aligned 16S dataset consisted of 524 bp, of which 433 were invariant sites, 72 were phylogenetically informative, and 19 were autapomorphies.

MP, ML, and NJ analyses produced congruent trees, and the GARLI ML phylogeny is presented in Fig. 2 A and B and 3A and B.

Both 16S and COI phylogenies identify five distinct monophyletic clades of vestimentiferans in the Gulf of Mexico. Four of the clades represent single morphospecies, *S. jonesi*, *E. laminata*, *Lamellibrachia* sp. 2, and escarpiid sp. nov., from the upper slope. However, the fifth clade includes both *Lamellibrachia* sp. 1 from the collections in the deeper GOM and *L. luymesi* from the upper Louisiana slope sites. They were, therefore, considered a single species when within- and between-species distances for the 16S and COI datasets were estimated. Additionally, COI sequences of *E. laminata* did not differ from those of *E. spicata* and

<footnote>a Samples analyzed for this study are numbered and labeled as for Figs. 2 and 3. Sequences from Genbank are listed by names assigned in Genbank.</footnote>

<footnote>b Samples from the Gulf of Mexico are indicated by GOM followed by the abbreviation of their collection sites. VK826, GC185, GC233, GB425, GC234, and GC354 are all on the upper Louisiana slope at depths <800 m. The other GOM sites are at depths >900 m and are indicated on Fig. 1.</footnote>
<figure><img src="image_4.png" /><figcaption>**Fig. 2.** COI maximum likelihood (ML) tree. Outgroups are shown in italics, and bootstrap support above 50% (NJ 1000 replicates) is indicated below each node. All new sequences are preceded by a number, followed by the abbreviation for the seep site or lease block from which they were collected. VK=Biosca Knoll, BH=Bush Hill, and BP=Brine Pool. Those sites, together with GB425, GC234, and GC354, are from the upper Louisiana slope of the GOM (< 800 m depth). All other lease blocks are on the lower slope (Fig. 1).</figcaption></figure>

**Table 2**

16 S Between- and within-species (in bold, on diagonal) *p* distances for the 16 S gene of the GOM species.

<table><thead><tr><th></th><th>[1] (%)</th><th>[2] (%)</th><th>[3] (%)</th><th>[4] (%)</th><th>[5] (%)</th></tr></thead><tbody><tr><td>[1] E. laminata</td><td><strong>0.10</strong></td><td></td><td></td><td></td><td></td></tr><tr><td>[2] L. luymesi/ sp. 1</td><td>9.60</td><td><strong>0.00</strong></td><td></td><td></td><td></td></tr><tr><td>[3] L. sp. 2</td><td>9.00</td><td>2.20</td><td><strong>0.10</strong></td><td></td><td></td></tr><tr><td>[4] S. jonesi</td><td>2.00</td><td>8.40</td><td>8.10</td><td><strong>0.00</strong></td><td></td></tr><tr><td>[5] Escarpid sp. new</td><td>3.50</td><td>8.30</td><td>8.90</td><td>3.70</td><td><strong>0.00</strong></td></tr></tbody></table>

**Table 3**

Between- and within-species (in bold, on diagonal) *p* distances for the COI gene.

<table><thead><tr><th></th><th>[1] (%)</th><th>[2] (%)</th><th>[3] (%)</th><th>[4] (%)</th><th>[5] (%)</th></tr></thead><tbody><tr><td>[1] E. laminata/southwardae/spicata</td><td><strong>0.9</strong></td><td></td><td></td><td></td><td></td></tr><tr><td>[2] L. luymesi/ sp. 1</td><td>13.7</td><td><strong>0.4</strong></td><td></td><td></td><td></td></tr><tr><td>[3] L. sp. 2</td><td>13.8</td><td>2.8</td><td><strong>0.3</strong></td><td></td><td></td></tr><tr><td>[4] S. jonesi</td><td>9.7</td><td>14.2</td><td>14.4</td><td><strong>0.3</strong></td><td></td></tr><tr><td>[5] Escarpid sp. new</td><td>7.1</td><td>14.8</td><td>14.6</td><td>7.1</td><td><strong>0.0</strong></td></tr></tbody></table>

*E. southwardae* from the East Pacific and East Atlantic, respectively. We were unable to obtain 16S sequences for *E. spicata* or *E. southwardae*.

Estimates of within- and between-species diversity (*p*) for both genes are shown in Tables 2 and 3. Within a species, *p* distances range from 0% to 0.1% for 16S and 0% to 0.9% for the more variable

COI. The very low values for the undescribed escarpiid may reflect the small number of individuals of this species analyzed (*n*=3 for COI and *n*=2 for 16S).

## 4. Discussion

### 4.1. Distribution of vestimentiferan species in the Gulf of Mexico and relation to other seep species

Vestimentiferans have been collected from both hydrothermal vent and cold-seep sites. The vent and seep species fall into two different clades. However, it should be noted that “seep species” are sometimes found in sedimented hydrothermal vent areas with low levels of diffuse flow, and that “cold-seep” fluids may have temperatures elevated over background (Black et al., 1998; Kojima et al., 1997; MacDonald et al., 2000; Joye et al., 2005); so this separation really reflects more aspects of their habitat than temperature alone. Vestimentiferans found at cold seeps worldwide can be further divided into two clades. One clade includes at least five named and three unnamed species in the genus *Lamellibrachia*. The other clade includes three named species in the genus *Escarpia*, *S. jonesi*, *Paraescarpia echinospica*, and a rarely collected species (escarpiid sp. nov.) from the shallow GOM. Although *Arcovestia* seems basal to the *Lamellibrachia* clade (Fig. 2B), this position is not well supported.

Three species in the escarpiid clade of seep vestimentiferans are found in the GOM: *S. jonesi* has been collected from numerous sites,
<figure><img src="image_5.png" /><figcaption>**Fig. 3.** 16S maximum likelihood (ML) tree. Outgroups are shown in italics and bootstrap support above 50% (NJ 1000 replicates) is indicated below each node. Sample identifications and abbreviations are as in Fig. 2.</figcaption></figure>

ranging in depth from 500 to 950 m; escarpid sp. nov. from two sites ranging in depth from 600 to 640 m, where it co-occurs with *S. jonesi* (although it has been reported also from GC234 at 525 m; see Cordes et al., 2003); and *E. laminata* from 950 to 3200 m depth. *S. jonesi* and *E. laminata* co-occurred at only one site, GB647, at a depth of 950 m. The undescribed escarpid differs morphologically from *S. jonesi*, as it lacks the curl of the ventral vestimental fold that is a defining character of the genus *Seepiophila* (Gardiner et al., 2001). Additionally, the obturacular process of the undescribed escarpid forms a spike, whereas it is flat in *S. jonesi* and barely protrudes from the top of the obturaculum.

Both the COI and 16S phylogenetic trees distinguish these three species and place them within the escarpid clade of seep vestimentiferans (Figs. 2 and 3). Both the 16S tree and the 16S *p* distance matrix suggest *E. laminata* is more closely related to *S. jonesi* (between-species uncorrected *p*≈2%) than to the undescribed escarpid (between-species uncorrected *p*≈3.50%). However, the COI tree groups the undescribed escarpid with the described *Escarpia* spp. The bootstrap value based on COI data supporting this clade is low (61%), and the grouping observed for the 16S dataset has a bootstrap below 50%. Neither tree allows us to state clearly whether this new escapiid is more closely related to *Escarpia*, *Paraescarpia*, or *Seepiophila*.

As previously noted by other authors, COI does not separate *Escarpia southwardae*, *E. spicata*, and *E. laminata*, respectively, from cold seeps on the west coast of Africa in the eastern Atlantic, Guaymas Basin, off the coast of Mexico, and the GOM (Black et al., 1998). Also, there is very little to no intra-clade diversity within this group (Table 3). This result may indicate that those three nominal species represent a single genealogical species with a surprisingly wide geographic distribution and variable morphology. However, this assumption would require a high level of gene flow between quite distant localities, especially since the closing of the Isthmus of Panama 3.5 million years ago, followed the closing of the deep sea exchange 10 million years ago (Burton et al., 1997). This level of genetic exchange over these distances seems quite unlikely, considering what is known about larval development times for vestimentiferans (Marsh et al. 2001, Young et al., 1996). Although the life span of *Escarpia* larvae has not been determined, the larval life span of the vent species *Riftia pachyptila* is estimated at about three weeks (Marsh et al., 2001)

and the larval life span of the seep vestimentiferan *L. luymesi* is estimated to be about one month (Young et al., 1996). Tyler and Young (1999) estimate that the maximal dispersal distances for these species are on the order of 60 km per generation, which is unlikely to support the level of genetic mixing necessary to maintain genetic homogeneity among the three described species of *Escarpia* from such widely separated geographic locations. It is possible, however, that undiscovered seeps around South America could connect all of these species.

The lack of fixed COI differences within *Escarpia spicata*, *E. laminata*, and *E. southwardae* could also be due to different rates of evolution of the COI gene in different taxa. COI has been used for higher level phylogenetic reconstructions in other groups of annelids (Halanych and Janosik, 2006) and has been adopted as an appropriate gene for the “barcode of life” for animals in general by the barcode of life initiative (BOLI; http://www.dnabarcodes.org/). However, the fact that COI fails to identify morphologically distinct populations of *Escarpia* from such widely separated areas implies that in this clade the mutation rate may be considerably slower than in other lineages. Slower rates of evolution in the mitochondrial DNA have been recognized in some other groups, such as the cnidarian class Anthozoa, where this phenomenon has been linked to an especially efficient repair system of their mitochondrial DNA (France and Hoover, 2002; Pont-Kingdon et al., 1998); however, no evidence of a similar system has been found in vestimentiferan mitochondrial DNA. Seep vestimentiferans can also be extremely long-lived (Bergquist et al., 2000; Cordes et al., 2007a), which may contribute to a slower rate of change of mitochondrial DNA (see for example Nabholz et al. (2008) for a consideration of longevity effects on mitochondrial rates of evolution in vertebrates).

In the COI dataset, the *Lamellibrachia* clade is divided into eight distinct groups that represent presumptive species, including five basal species (*L. juni*, *L. barhami*, *L. satsuma*, *L. sp*. Japan, and *L. sp*. West Pacific), all of which are from the Pacific Ocean and four of which are from the western Pacific. This observation is consistent with the hypothesis that the genus *Lamellibrachia* originated in the Pacific, likely the western Pacific, and subsequently radiated to the eastern Pacific, the Atlantic, and the GOM.

Three morphological species of *Lamellibrachia* were identified in collections from the GOM: *L. luymesi*, from the upper slope at
between about 400 m and 800 m; *L. sp.* 1, from 950 to 2320 m; and *L. sp.* 2, from 1175 to 2320 m. *L. luymesi* and *L. sp.* 1 have a similar number of sheath lamellae, but the deep-water *L. sp.* 1 generally has more gill lamellae, ranging between 21 and 27 in the 28 individuals examined, whereas the shallow-water *L. luymesi* has between 15 and 22 gill lamellae in the 20 individuals examined for the species description. The morphological character that allowed rapid identification of animals aboardship was the relatively short and fat vestimentum of *L. sp.* 1. The ratio of length to width of the vestimentum of *L. sp.* 1 ranges from 2.4 to 4.7 and from 6.2 to 16.4 in *L. luymesi*. *L. sp.* 2 has a similar number of sheath and gill lamellae as *L. sp.* 1, and the vestimentum length to width ratio tends to be shorter (1.9 to 3). The most distinct field character for *L. sp.* 2 is the lack of a ventral vestimental fold, which is present on *L. sp.* 1.

Despite morphological characters that distinguish the three GOM *Lamellibrachia* presumptive species, only either the COI or the 16S phylogenetic trees resolved two of them. Specifically, both genes failed to separate *L. luymesi* from the shallow GOM and *L. sp.* 1 from the deeper GOM sites. This lack of genetic differences between individuals that span such a wide depth range is unusual (Chase et al., 1998; Zardus et al., 2006) and surprising, given the morphological differences. Both 16S and COI genes consistently identify *Lamellibrachia* sp. 2 as a separate clade, sister to the *L. luymesi/L. sp.* 1 clade.

There were no apparent geographic distributional patterns that were independent of depth for the seep vestimentiferans in the Gulf of Mexico. The common species present on the upper Louisiana slope (*L. luymesi* and *S. jonesi*) have been found at both the eastern-most and western-most sites where we have collected vestimentiferans. *E. laminata* from the lower slope ranges from the Alaminos Canyon sites, our most westerly collection sites for this study, to the Florida Escarpment in the eastern GOM (Cordes et al., 2009; McMullin et al., 2003). Both of the *Lamellibrachia* spp. found at the deeper sites occurred over the entire E-W range of sites within their depth range (from the Alaminos Canyon sites in the west to AT340 in the east).

## 4.2. Within-species diversity of the GOM vestimentiferans

Tables 2 and 3 report within- and between-species *p* distance calculated for the GOM genetic species. In most cases, within-species diversity for both 16S and in the COI genes is strikingly low, a finding that is in contrast to previous studies on deep-sea mollusks and echinoderms, where large amounts of genetic variation were observed over small distances (Chase et al., 1998; Howell et al., 2004; Quattro et al., 2001). However, large-scale studies indicate that low within-species genetic variation may be typical of deep-sea organisms (Bisol et al., 1984) and even suggest that it may decrease with increase in depth (France and Kocher, 1996). Genetic variation has been suggested to be an important feature of the genome of an organism that allows it to adapt to a changing environment (Powers et al., 1991). Organisms that live in the deep sea may experience a long-term stable environment, resulting in low levels of within-species genetic diversity. Alternatively, low within-species genetic diversity may be the result of fewer replication errors, more efficient repair in the germ line, or repeated population bottlenecks.

*E. laminata*, *E. spicata*, and *E. southwardae* clade and *L. luymesi* sp. 1 and *L. sp.* 2 have a moderate degree of intra-specific diversity (Figs. 2 and 3). However, as with all of the GOM vestimentiferans analyzed, none of the within-species clades grouped by specific geographic locations or depth. A similar pattern was found in the seep mussel *Bathymodiolus childressi*, which, based on markers ranging from microsatellites to mitochondrial genes, has a panmictic population in the GOM ranging across 550 km east to west and from 540 to 2200 m depth (Carney et al., 2006; Cordes

et al., 2007b). In contrast, genetic breaks and barriers that restrict gene flow were identified in both hydrothermal vent vestimentiferans and mussels along the East Pacific Rise (EPR). Specifically, Won et al. (2003) used COI sequences to identify two highly divergent clades on the EPR on the two sides of the Easter Island Microplate. Similarly, Hurtado et al. (2004) used COI sequences to identify several geographic breaks and barriers that restrict gene flow in three genera of annelids along the EPR, including two species of vestimentiferan (*Riftia pachypitla* and *Tevnia jerichonana*).

## 5. Summary

In this study, our primary goals were to identify and characterize the distributions of vestimentiferans at seep sites covering a wide geographic and depth range in the Gulf of Mexico and to investigate their relationship to other seep vestimentiferan species, using phylogenetic analysis of mitochondrial gene sequences. Although the genetic analyses confirmed identification of most of the morphological species during collections, we also identified an unexpected discrepancy between the morphospecies identified during the collections and genealogical species identified using the mitochondrial genes COI and 16S. Using morphological characters, we identified two new species of *Lamellibrachia* (spp. 1 and 2). However, neither COI nor 16S distinguished the deeper occurring morphospecies *L. sp.* 1 from *L. luymesi*, the common *Lamellibrachia* species on the upper Louisiana slope. Our molecular genetic analyses confirm the presence of three vestimentiferan species within the escarpid clade in the Gulf of Mexico. However, since COI also does not differentiate between *E. laminata* found in the Gulf of Mexico and the other described *Escarpia* species off the coast of Africa or in the eastern Pacific Ocean, we suggest that COI or 16S genes may not reliably distinguish closely related species of long-lived seep vestimentiferans. We are currently evaluating the usefulness of several nuclear genes to clarify the relationships among the named species of *Escarpia* and the *Lamellibrachia* species in the Gulf of Mexico.

## Acknowledgments

This work was funded by a subcontract to Bureau of Ocean Energy Management, Regulation and Enforcement (BOEMRE) contract #1435-01-05-39187, "Investigations of Chemosynthetic Communities on the Lower Continental Slope of the Gulf of Mexico (Chemo III)," with vessel and submergence facilities support provided by National Oceanic and Atmospheric Administration's Office of Ocean Exploration and Research. This study would not have been possible without the support and expertise of the captains, crews, and expedition leaders of the Research Vessel *Atlantis*, the DSV ALVIN, the NOAA ship *Ronald Brown*, and the ROV JASON II. The authors are grateful to Erik Cordes and Erin Becker for help at sea, to Stephanie Lessard-Pilon, Erin Becker, and Meredith Cole Patterson for help in the laboratory, to Erin McMullin for sharing DNA and expertise, and to A. Faucci and ChEss Siboglinidae Workshop participants for suggestions and helpful discussion.

## References

* Bergquist, D.C., Urcuyo, I.A., Fisher, C.R., 2002. Establishment and persistence of seep vestimentiferan aggregations on the upper Louisiana slope of the Gulf of Mexico. Marine Ecology—Progress Series 241, 89–98.

* Bergquist, D.C., Williams, F.M., Fisher, C.R., 2000. Longevity record for deep-sea invertebrate. Nature 403 (6769), 499–500.

* Bisol, P.M., Costa, R., Sibuet, M., 1984. Ecological and genetic survey on two deep-sea holothurians—benthogone-rosea and benthodytes-typica. Marine Ecology—Progress Series 15 (3), 275–281.
Black, M.B., Trivedi, A., Maas, P.A.Y., Lutz, R.A., Vrijenhoek, R.C., 1998. Population genetics and biogeography of vestimentiferan tube worms. Deep-Sea Research II: Topical Studies in Oceanography 45, 1-3.

Burton, K.W., Ling, H.F., Onions, R.K., 1997. Closure of the Central American Isthmus and its effect on deep-water formation in the North Atlantic. Nature 386 (6623), 382-385.

Carney, S.L., Formica, M.I., Divatia, H., Nelson, K., Fisher, C.R., Schaeffer, S.W., 2006. Population structure of the mussel "Bathymodiolus" *childressi* from Gulf of Mexico hydrocarbon seeps. Deep-Sea Research I: Oceanographic Research Papers 53 (6), 1061-1072.

Chase, M.R., Etter, R.J., Rex, M.A., Quattro, J.M., 1998. Bathymetric patterns of genetic variation in a deep-sea protobranch bivalve, *Deminucula atacellana*. Marine Biology 131 (2), 301-308.

Cordes, E.E., Bergquist, D.C., Fisher, C.R., 2009. Macro-ecology of Gulf of Mexico cold seeps. Annual Review of Marine Science 1, 143-168.

Cordes, E.E., Bergquist, D.C., Redding, M.L., Fisher, C.R., 2007a. Patterns of growth in cold-seep vestimentiferans including *Seepiophila jonesi*: a second species of long-lived tubeworm. Marine Ecology-An Evolutionary Perspective 28 (1), 160-168.

Cordes, E.E., Becker, E.L., Hourdez, S., Fisher, C.R., 2010. Influence of foundation species, depth, and location on diversity and community composition at Gulf of Mexico lower-slope cold seeps. Deep-Sea Research II: Topical Studies in Oceanography 57 (21-23), 1870-1881.

Cordes, E.E., Carney, S.L., Hourdez, S., Carney, R.S., Brooks, J.M., Fisher, C.R., 2007b. Cold seeps of the deep Gulf of Mexico: Community structure and biogeographic comparisons to Atlantic equatorial belt seep communities. Deep-Sea Research I: Oceanographic Research Papers 54 (4), 637-653.

Cordes, E.E., Bergquist, D.C., Shea, K., Fisher, C.R., 2003. Hydrogen sulfide demand of long-lived vestimentiferan tube worm aggregations modifies the chemical environment at deep-sea hydrocarbon seeps. Ecology Letters 6 (2003), 212-219.

Doyle, J.J., Doyle, J.L., 1987. A rapid DNA isolation procedure for small quantities of fresh leaf tissue. Phytochemical Bulletin 19, 11-15.

Felsenstein, J., 1985. Confidence limits on phylogenies: an approach using the bootstrap. Evolution 39 (4), 783-791.

Folmer, O., Black, M., Hoeh, W., Lutz, R., Vrijenhoek, R., 1994. DNA primers for amplification of mitochondrial cytochrome c oxidase subunit I from diverse metazoan invertebrates. Molecular Marine Biology and Biotechnology 3 (5), 294-299.

France, S.C., Hoover, L.L., 2002. DNA sequences of the mitochondrial COI gene have low levels of divergence among deep-sea octocorals (Cnidaria: Anthozoa). Hydrobiologia 471, 149-155.

France, S.C., Kocher, T.D., 1996. Geographic and bathymetric patterns of mitochondrial 16S rRNA sequence divergence among deepsea amphipods, *Eurythenes gryllus*. Marine Biology 126 (4), 633-643.

Gardiner, S.L., McMullin, E., Fisher, C.R., 2001. *Seepiophila jonesi*, a new genus and species of vestimentiferan tube worm (Annelida: Pogonophora) from hydrocarbon seep communities in the Gulf of Mexico. Proceedings of the Biological Society of Washington 114 (3), 694-707.

Guindon, S., Gascuel, O., 2003. A simple, fast, and accurate algorithm to estimate large phylogenies by maximum likelihood. Systematic Biology 52 (5), 696-704.

Halanych, K.M., Feldman, R.A., Vrijenhoek, R.C., 2001. Molecular evidence that *Sclerolinum brattstromi* is closely related to vestimentiferans, not to *Frenulate pogonophorans* (Siboglinidae: Annelida). Biological Bulletin 201 (1), 65-75.

Halanych, K.M., Janosik, A.M., 2006. A review of molecular markers used for annelid phylogenetics. Integrative and Comparative Biology 46 (4), 533-543.

Howell, K.L., Rogers, A.D., Tyler, P.A., Billett, D.S.M., 2004. Reproductive isolation among morphotypes of the Atlantic seastar species *Zoroaster fulgens* (Asteroidea: Echinodermata). Marine Biology 144 (5), 977-984.

Hurtado, L.A., Lutz, R.A., Vrijenhoek, R.C., 2004. Distinct patterns of genetic differentiation among annelids of eastern Pacific hydrothermal vents. Molecular Ecology 13 (9), 2603-2615.

Joye, S.B., MacDonald, I.R., Montoya, J.P., Peccini, M., 2005. Geophysical and geochemical signatures of Gulf of Mexico seafloor brines. Biogeosciences 2, 1-15.

Kojima, S., Ohta, S., Yamamoto, T., Miura, T., Fujiwara, Y., Fujikura, K., Hashimoto, J., 2002. Molecular taxonomy of vestimentiferans of the western Pacific and their phylogenetic relationship to species of the eastern Pacific-II-Families Escarpidae and Arcovestiidae. Marine Biology 141 (1), 57-64.

Kojima, S., Segawa, R., Hashimoto, J., Ohta, S., 1997. Molecular phylogeny of vestimentiferans collected around Japan, revealed by the nucleotide sequences of mitochondrial DNA. Marine Biology 127 (3), 507-513.

Kojima, S., Segawa, R., Kobayashi, T., Hashimoto, T., Fujikura, K., Hashimoto, J., Ohta, S., 1995. Phylogenetic relationships among species of *Calypトgena*

(bivalvia, vesicomyidae) collected around Japan revealed by nucleotide sequences of mitochondrial genes. Marine Biology 122 (3), 401-407.

MacDonald, I.R., Buthman, D., Sager, W.W., Peccini, M.B., Guinasso Jr., N.R., 2000. Pulsed oil discharge from a mud volcano. Geology 28 (10), 907-910.

Maddison, D.R., Maddison, W.P., 2000. MacClade 4: Analysis of Phylogeny and Character Evolution. Sinauer Associates Inc., Sunderland, MA, USA.

Marsh, A.G., Mullineaux, L.S., Young, C.M., Manahan, D.T., 2001. Larval dispersal potential of the tubeworm *Riftia pachyptila* at deep-sea hydrothermal vents. Nature 411 (6833), 77-80.

McMullin, E.R., Hourdez, S., Schaeffer, S.W., Fisher, C.R., 2003. Phylogeny and biogeography of deep sea vestimentiferan tubeworms and their bacterial symbionts. Symbiosis 34 (1), 1-41.

Mironov, A.N., 2000. New taxa of stalked crinoids from the suborder *Bourguetiocrinina* (Echinodermata, Crinoidea). Zoologichesky Zhurnal 79 (6), 712-728.

Miura, T., Nedachi, M., Hashimoto, A., 2002. Sulphur sources for chemoautotrophic nutrition of shallow water vestimentiferan tubeworms in Kagoshima Bay. Journal of the Marine Biological Association of the United Kingdom 82 (4), 537-540.

Nabholz, B., Glemin, S., Galtier, N., 2008. Strong variations of mitochondrial mutation rate across mammals - the longevity hypothesis. Molecular Biology and Evolution 25 (1), 120-130.

Palumbi, S.R., Warner, R.R., 2003. Ecology. Why gobies are like Hobbits. Science 299 (5603), 51-52.

Pont-Kingdon, G., Okada, N.A., Macfarlane, J.L., Beagley, C.T., Watkins-Sims, C.D., Cavalier-Smith, T., Clark-Walker, G.D., Wolstenholme, D.R., 1998. Mitochondrial DNA of the coral *Sarcophyton glaucum* contains a gene for a homologue of bacterial MutS: a possible case of gene transfer from the nucleus to the mitochondrion. Journal of Molecular Evolution 46 (4), 419-431.

Posada, D., 2003. Using MODELTEST and PAUP* to select a model of nucleotide substitution. Current Protocols for Bioinformatics, Unit 6, 5 Chapter 6.

Posada, D., Crandall, K.A., 1998. MODELTEST: testing the model of DNA substitution. Bioinformatics 14 (9), 817-818.

Powers, D.A., Lauerman, T., Crawford, D., DiMichele, L., 1991. Genetic mechanisms for adapting to a changing environment. Annual Review of Genetics 25, 629-659.

Quattro, J.M., Chase, M.R., Rex, M.A., Greig, T.W., Etter, R.J., 2001. Extreme mitochondrial DNA divergence within populations of the deep-sea gastropod *Frigidovalvania brychia*. Marine Biology 139 (6), 1107-1113.

Rouse, G., 2001. A cladistic analysis of *Siboglinidae caullery*, 1914 (Polychaeta, Annelida): formerly the phyla pogonophora and vestimentifera. Zoological Journal of the Linnean Society 132, 55-80.

Saitou, N., Nei, M., 1987. The neighbor-joining method: a new method for reconstructing phylogenetic trees. Molecular and Biological Evolution 4, 406-425.

Tamura, K., Dudley, J., Nei, M., Kumar, S., 2007. MEGA4: molecular evolutionary genetics analysis (MEGA) software version 4.0. Molecular Biology and Evolution 24 (8), 1596-1599.

Thompson, J.D., Gibson, T.J., Higgins, D.G., 2002. Multiple sequence alignment using ClustalW and ClustalX. Current Protocols in Bioinformatics, Chapter 2, Unit 23.

Thornhill, D.J., Mahon, A.R., Norenburg, J.L., Halanych, K.M., 2008. Open-ocean barriers to dispersal: a test case with the Antarctic Polar Front and the ribbon worm *Parborlasia corrugatus* (Nemertea: Lineidae). Molecular Ecology 17 (23), 5104-5117.

Tyler, P.A., Young, C.M., 1999. Reproduction and dispersal at vents and cold seeps. Journal of the Marine Biological Association of the United Kingdom 79, 193-208.

van der Land, J., Narrevang, A., 1975. The systematic position of Lamellibrachia [sic] (annelida: vestimentifera). In: Norrevang, A. (Ed.), The Phylogeny and Systematic Position of Pogonophora, pp. 86-101.

Wilgenbusch, J.C., Swofford, D., 2003. Inferring evolutionary trees with PAUP*. Current Protocols in Bioinformatics, Chapter 6, Unit 64.

Won, Y., Young, C.R., Lutz, R.A., Vrijenhoek, R.C., 2003. Dispersal barriers and isolation among deep-sea mussel populations (mytilidae: *Bathymodiolus*) from eastern Pacific hydrothermal vents. Molecular Ecology 12 (1), 169-184.

Young, C.M., Vazquez, E., Metaxas, A., Tyler, P.A., 1996. Embryology of vestimentiferan tube worms from deep-sea methane/sulphide seeps. Nature 381 (6582), 514-516.

Zardus, J.D., Etter, R.J., Chase, M.R., Rex, M.A., Boyle, E.E., 2006. Bathymetric and geographic population structure in the pan-Atlantic deep-sea bivalve *Deminucula atacellana* (Schenck, 1939). Molecular Ecology 15 (3), 639-651.

Zwickl, D.J., 2006. Genetic algorithm approaches for the pylogenetic analysis of large biological sequence datasets under the maximum likelihood criterion. [www.bioutexas.edu/faculty/antisense/garli/Garli.html] Ph.D. dissertation, The University of Texas at Austin, TX, USA.