DESCRIPTION OF TWO NEW SPECIES OF HYALELLA

JOURNAL OF CRUSTACEAN BIOLOGY, 35(6), 814-829, 2015 DESCRIPTION OF TWO NEW SPECIES OF HYALELLA (AMPHIPODA: HYALELLIDAE) FROM EASTERN NORTH AMERICA WITH A REVISED KEY TO NORTH AMERICAN MEMBERS OF THE GENUS David J. Soucek 1,, Eric A. Lazo-Wasem 2, Christopher A. Taylor 1, and Kaley M. Major 3 1 Illinois Natural History Survey, 1816 S. Oak Street, Champaign, IL 61820, USA 2 Peabody Museum of Natural History, Yale University, 170 Whitney Avenue, New Haven, CT 06520, USA 3 University of Massachusetts-Boston, School for the Environment, 100 Morrissey Boulevard, Boston, MA 02125, USA ABSTRACT Since the 1990s, it has been presumed that different populations/size classes of the freshwater amphipod Hyalella azteca (Saussure, 1858) were different species, and a multitude of genetic evidence was later produced to confirm that it is in fact a cryptic species complex. However, no new species of Hyalella have been described from the eastern United States or Canada since then. Herein we describe two new species, Hyalella (Hyalella) spinicauda and Hyalella (Hyalella) wellborni, collected from Illinois, Indiana, Michigan, Wisconsin, and Texas, USA, and Ontario, Canada, based on morphological characters but informed by genetic analyses. In addition, we provide an updated key to the described species of Hyalella in North America and the Caribbean. KEY WORDS: Amphipoda, Hyalella, new species DOI: 10.1163/1937240X-00002373 INTRODUCTION The freshwater amphipod Hyalella azteca (Saussure, 1858) is found in many different lentic and lotic habitats across North and Central America and is considered to be one of the most widely dispersed invertebrates in North America (Witt and Hebert, 2000). The species was originally described as Amphithoe azteca in 1858 from material collected in Vera Cruz and Mexico City, Mexico (Saussure, 1858). Gonzalez and Watling (2002) redescribed H. azteca in more detail using the original type material collected by Saussure, and provide a thorough history of the various descriptions and synonymies, including Hyalella knickerbockeri (Bate, 1862), Lockingtonia fluvialis Harford, 1877, Hyalella dentata Smith, 1874, Hyalella faxoni Stebbing, 1903, and Hyalella ornata Pearse, 1911, into Hyalella azteca. However, Strong (1972) observed distinct differences in life history characteristics among different populations of H. azteca, and Wellborn (1993, 1994a, b, 1995a, b) and Wellborn et al. (2005) characterized distinct size classes of this species among populations in Michigan and Oklahoma. Wellborn (1994a) suggested that the two size classes might represent different species (with each size class potentially consisting of several species), attributing the size difference to presence or absence of fish predators. A preliminary morphometric analysis further supported separate species status (Zakharia, 1994). Subsequently, a number of genetic studies were published supporting the suggestion that H. azteca as currently described is in reality, a species complex (Duan et al., 1997; Hogg et al., 1998; McPeek and Wellborn, 1998; Thomas et al., 1998; Witt et al., 2006; Major et al., 2013). The diversity of described species of Hyalella has greatly increased in recent years, particularly in South America where greater than 30 species are known from Brazil, Argentina, and Chile alone (Gonzalez, 2003; Santos et al., 2008; Rodriques et al., 2014). In North America and the Caribbean region, eleven species of Hyalella are currently recognized (Baldinger, 2004; Marrón-Becerra et al., 2014). Many of the species are restricted to specific habitats; for example, H. montezuma Cole and Watkins, 1977 is found exclusively in Montezuma s Well in Arizona, H. cenotensis Marrón-Becerra, Hermoso-Salazar, and Solís-Weiss, 2014 is found in the sinkhole, Cenote Aktun-Ha, in Quintana Roo, Mexico, and H. meraspinosa Baldinger, 2004 is known only from its type locality in Ash Springs, Nevada. Hyalella texana Stevenson and Peden, 1973 has been found in several locations within the Edwards Plateau of Texas (Levine, 1999; Stevenson and Peden, 1973). The remaining species of Hyalella in North America are either found on the Caribbean island of Guadeloupe (H. caribbeana Bousfield, 1996; H. squamosa Mateus and Mateus, 1990) or in the desert southwest of the United States (H. inermis Smith, 1875; H. muerta and H. sandra Baldinger, Shepard, and Threloff, 2000; H. longicornis Bousfield, 1996). Despite the long held suspicion that different populations/size classes in Michigan were different species (Wellborn, 1993), and the multitude of genetic evidence to support this suspicion, no new species of Hyalella have been described from the eastern United States or Canada. Herein we describe two Corresponding author; e-mail: soucek@illinois.edu The Crustacean Society, 2015. Published by Brill NV, Leiden DOI:10.1163/1937240X-00002373

SOUCEK ET AL.: TWO NEW SPECIES OF NORTH AMERICAN HYALELLA 815 new species of Hyalella collected from Illinois, Indiana, Michigan, Wisconsin, and Texas, USA, and Ontario, Canada using morphological characters not examined or utilized in previous analyses or keys (Baldinger, 2004). MATERIALS AND METHODS Most specimens used in the present descriptions were collected from field sites between December 2009 and September 2011, but specimens from Indiana and Texas were from the Illinois Natural History Survey Crustacean Collection (INHS) and had morphological characteristics that matched the more recent field collections. In some cases, laboratory-reared descendants of field collected specimens were used for examination of characters. Specimens collected from the field were preserved in 95% ethanol so material could be used in genetic analyses (Major et al., 2013) in addition to morphological studies. Specimens of both species were found at four of the sites: Clear Pond, IL, USA; Ives Lake, MI, USA; Pine River, MI, USA; and Tittabawassee River, MI, USA (Table 1). We examined the male specimen of Amphithoe azteca, originally collected by Saussure (1858), re-described and designated as the H. azteca lectotype by Gonzalez and Watling (2002), and loaned to us by Muséum d histoire naturelle de la Ville de Genève (no catalog number assigned), and we compared it to specimens from our laboratory culture. Laboratory culture specimens originated from a commercial source (Aquatic Research Organisms, Inc., Hampton, NH). We determined that these specimens from our laboratory are the same species as the lectotype, based on morphology of the maxilla 1, gnathopod 2, pereiopod 7, uropod 3, and telson. This species is genetically identified as the US Lab clade by Major et al. (2013), and complete cytochrome oxidase subunit 1 (COI) sequences can be found at Genbank accession Nos JX446307 and JX446308. It is referred to as the OK-L clade by Wellborn and Broughton (2008). Specimens of H. azteca from our laboratory culture were used to create drawings for comparison to the two new species described here. We also compared the two new species described here to the type specimens of H. dentata (Smith, 1874) (YPM IZ 009143). Most morphological features were examined under a dissecting microscope, but mouthparts were examined using a compound microscope. Illustrations were made by photographing stained appendages using a Zeiss Stemi 2000-CS microscope fitted with a Q- Imaging Micropublisher 5.0 RTV digital camera, and creating line drawings using Adobe Illustrator CS5 (version 15.1.0; 2011 Adobe Systems). Drawings are of male and females specimens; all are deposited in the Illinois Natural History Survey Crustacean Collection, Champaign, Illinois, or Yale University Peabody Museum (YPM), New Haven, CT, USA. SYSTEMATICS Hyalella (Hyalella) spinicauda Soucek and Lazo-Wasem n. sp. (Figs. 1-5, 10) Diagnosis. Eye pigmented. Maxilla 1, inner plate with 2 pappose setae. Gnathopod 2 (males), carpus posterior lobe free portion approximately equal in length to width of merus; palm oblique, with a distinct angled notch (sometimes visible only under high power). Pereiopod 7, basis posterior lobe ventral margin usually without dentation, may have one or two short, fine setae, but no large spines. Uropod 3, ramus slightly shorter than peduncle; longest peduncle seta greater than half the length of ramus; ramus setae slightly less robust than peduncle setae. Telson, rounded, terminal setae stout and widely spaced; telson terminal setae and those on ramus of U3 similar in stoutness. Description of Holotype Male. Pleonites 1 and 2 with dorsoposterior mucronations. Coxae 1-4 with minute setae on distal margin, coxa 4 excavated posteriorly and larger than 1-3; coxa 5 with distinct anterior and posterior lobes; coxa 6 with a minute anterior lobe; coxa 7 reduced. Coxal gills (on pereionites 2-6) larger than sternal gills (on pereionites 3-7). Head, cuboidal, shorter in length than pereionites 1 and 2 combined (Fig. 1). Eye pigmented, generally ovoid, close to dorsal and anterior margins of head, diameter at longest point greater than the width of the basal segment of the peduncle of antenna 1. Antenna 2 slightly longer than Table 1. Collection sites for two new species of Hyalella. Type locality. a Sample from Illinois Natural History Survey Crustacean Collection. b Latitude and longitude are approximations due to insufficient information. Site County State/Province Latitude (N) Longitude (W) Hyalella spinicauda Clear Pond Vermilion IL, USA 40.1381 87.7417 Ives Lake Marquette MI, USA 46.8437 87.8548 Pine River Marquette MI, USA 46.8831 87.8687 Middle Pine Lake Marquette MI, USA 46.8699 87.8559 Tittabawassee River Saginaw MI, USA 43.4812 84.0924 Winnebago Lake Calumet WI, USA 44.1126 88.3253 La Crosse River La Crosse WI, USA 43.9008 90.9901 Valens Reservoir Wentworth ON, CAN 43.3867 80.1333 Hyalella wellborni Japan House Pond Champaign IL, USA 40.0932 88.2168 E. Fork Embarras River Champaign IL, USA 39.9451 88.1231 Clear Pond Vermilion IL, USA 40.1381 87.7417 Flat Branch Coles IL, USA 39.5997 88.3214 Pope Branch Edgar IL, USA 39.8141 87.9273 Panther Creek Perry IL, USA 38.0235 89.3337 Indian Creek Jackson IL, USA 37.6557 89.1798 Eagle Creek Saline/Gallatin IL, USA 37.6558 88.3743 Miller s Pond a, b Porter IN, USA 41.6869 86.9895 Ives Lake Marquette MI, USA 46.8437 87.8548 Pine River Marquette MI, USA 46.8831 87.8687 Tittabawassee River Saginaw MI, USA 43.4812 84.0924 Galvan Creek a Atascosa TX, USA 29.0388 98.4855

816 Fig. 1. JOURNAL OF CRUSTACEAN BIOLOGY, VOL. 35, NO. 6, 2015 A, Hyalella spinicauda. INHS 14856, holotype male 5.76 mm. B, Hyalella wellborni. INHS 14859, holotype male 4.28 mm.

SOUCEK ET AL.: TWO NEW SPECIES OF NORTH AMERICAN HYALELLA 817 antenna 1; antenna 1 approx. 25% of body length, antenna 2 approx. 30% of body length. Upper lip (Fig. 2) rounded, anterior margin with many fine setae. Mandible, lacking a palp, but otherwise similar to basic mandible of Watling (1993); left, spine row with 3 plumose setae, molar normal; right, 2 plumose setae on spine row, molar normal, bearing a plumose accessory flagellum. Lower lip, outer lobes rounded with many fine setae on inner and anterior margins. Maxilla 1, inner plate narrow, with 2 apical plumose setae; outer plate with 9 stout, serrate and comb-like setae; palp vestigial, 1 article coming to an acute point. Maxilla 2, plates subequal in width; inner plate inner margin with 2 long plumose setae and many apical setae; outer plate with many long apical setae. Maxilliped, inner plates sub-rectangular, each with 3 strong apical teeth, inner and apical margins with plumose setae; outer plates similar in width to inner plates, the apical end ovoid, with setae on inner and apical margins; palp with 4 articles, articles 1 and 2 wider than long, article 2 inner margin setose, article 3 apical margin with relatively long setae, terminal article triangular, apical margin bearing a long robust spine and 2 or 3 shorter setae. Gnathopod 1 (Fig. 3) basis longer than wide, the distal end wider than the proximal; carpus longer than wide, longer than the propodus, with a long setae on anterior margin, and 9 long setae on posterior lobe; carpus wider than merus; propodus subrectangular, palm transverse with marginal and submarginal setae. Gnathopod 2 basis longer than wide; carpus subtriangular, much shorter than propodus, free portion of carpus posterior lobe approximately equal in length to width of merus; palm oblique, with a distinct angled notch (sometimes visible only under high power), posteroproximal corner produced, rounded, with several robust setae. Pereiopods 3 and 4 (Fig. 4) similar in size and shape, merus anterodistal lobes extending approx. 1/4 or less the length of respective carpus; pereiopod 5 distinctly shorter than 6 and 7 which are similar in length; Pereiopods 5-7 bases posterior margin expanded (more so in 5 and 7 than in 6), finely serrate; pereiopod 7 basis posterior lobe ventral margin without dentation, or setae. Pleopods, long and slender, all with 2 to 3 coupling hooks on peduncle. Uropod 1 (Fig. 5) longer than uropod 2. Uropod 1, peduncle longer than rami, which are similar in length; uropod 2, rami similar in length; uropod 3 extending slightly beyond uropod 2 peduncle distal margin, ramus slightly shorter than peduncle, ramus approximately as long as or slightly longer than longest seta of peduncle, ramus setae about as robust as peduncle setae. Telson, rounded, terminal setae stout and widely spaced; telson terminal setae and those on peduncle and ramus of U3 similar in stoutness. Allotype Female (Fig. 10). Similar in structure to male except for as follows. Antenna 1 approx. 29% of body length, antenna 2 approx. 36% of body length. Gnathopod 2 similar in structure to gnathopod 1 (which is similar to gnathopod 1 of males), propodus longer than carpus, longer than propodus of gnathopod 1. Setose oöstegites present on gnathopod 2 and pereiopods 3-5. Other Variations. In some adult males, pereiopod 7 basis posterior lobe ventral margin may have limited dentation, and one or two short, fine setae. Etymology. spinicauda = spine tailed; in reference to the robust terminal spines of the telson. Disposition of Types and Material Examined. The holotype and allotype (INHS 14856 and 14857) for H. spinicauda were collected from Clear Pond, Kickapoo State Park, IL (Table 1) and deposited in the INHS Crustacean Collection. Paratypes (INHS 14858) collected from Clear Pond (Table 1) were deposited in the INHS Crustacean Collection, and additional paratypes (YPM IZ 076754) collected from Valens Reservoir, Wentworth, ON, Canada (Table 1), were deposited in the Yale University Peabody Museum of Natural History. Notes on Habitat and Range. Individuals of this new species were collected from both wadable lentic and lotic habitats in Illinois, Michigan, and Wisconsin, USA, and Ontario, Canada (Table 1), but more fieldwork is needed to determine its full range. The type material was collected along the shore of Clear Pond, Kickapoo State Park, by sweeping submerged macrophytes with a d-frame net. Hyalella (Hyalella) wellborni Soucek and Lazo-Wasem, new species (Figs. 6-10) Diagnosis. Eye pigmented. Maxilla 1, inner plate with 2 pappose setae. Gnathopod 2 (males), carpus posterior lobe free portion approximately equal to or shorter than merus widths; palm strongly oblique, regular, with no distinct notch or truncated process. Pereiopod 7, basis posterior lobe ventral margin usually not dentate and without setae one or two dentations with very fine setae may be present, but no large spines. Uropod 3, ramus shorter than peduncle; longest peduncle seta nearly as long as ramus; ramus setae robust, as thick as peduncle setae. Telson, tapered to a rounded point, terminal setae closely set; terminal setae clearly finer than setae of uropod 3 ramus. Description of Holotype Male. Pleonites 1 and 2 with dorsoposterior mucronations. Coxae 1-4 with minute setae on distal margin, coxa 4 excavated posteriorly and larger than 1-3; coxa 5 with distinct anterior and posterior lobes; coxa 6 slightly reduced with a minute anterior lobe; coxa 7 reduced. Coxal gills (on pereionites 2-6) larger than sternal gills (on pereionites 3-7). Head, cuboidal, slightly shorter in length than pereionites 1 and 2 combined (Fig. 1). Eye pigmented, generally ovoid, close to dorsal and anterior margins of head, diameter at longest point greater than the width of antenna 1 peduncle basal segment. Antenna 2 slightly longer than antenna 1; antenna 1 approx. 33% of body length, antenna 2 approx. 40% of body length. Upper lip (Fig. 6), rounded, anterior margin with many fine setae. Mandible, lacking a palp, but otherwise similar to basic mandible of Watling (1993); right, incisor process 5-toothed, lacinia 4-toothed, spine row with 3 plumose setae, molar normal, triturative; left, lacinia with two large and numerous smaller teeth. Lower lip, outer lobes rounded with many fine setae on inner and anterior margins. Maxilla 1,

818 JOURNAL OF CRUSTACEAN BIOLOGY, VOL. 35, NO. 6, 2015 Fig. 2. Hyalella spinicauda. INHS 14856, holotype male 5.76 mm. UL, upper lip; LL, lower lip; Mxpd, maxilliped; ip, maxilliped inner plate; Mx1, maxilla 1; Mx2, maxilla 2; lmd, left mandible; rmd, right mandible.

SOUCEK ET AL.: TWO NEW SPECIES OF NORTH AMERICAN HYALELLA 819 Fig. 3. Hyalella spinicauda. INHS 14856, holotype male 5.76 mm. Gn1, gnathopod 1; Gn2, gnathopod 2. inner plate narrow, with 2 apical plumose setae; outer plate with 9 stout, serrate and comb-like setae; palp vestigial, 1 article with a small apical knob. Maxilla 2, plates subequal in width; inner plate inner margin with 2 long robust setae and many apical setae; outer plate with many long apical setae. Maxilliped, inner plates sub-rectangular, each with 3 strong apical teeth, inner and apical margins with plumose setae; outer plates similar in width to inner plates, apical end ovoid, with setae on inner and apical margins; palp with 4 articles, article 1 wider than long, article 2 inner margin setose, apical margin of article 3 with relatively long setae (some plumose), terminal article triangular, apical margin bearing a long robust spine and 2 or 3 shorter setae. Gnathopod 1 (Fig. 7), basis longer than wide, the distal end wider than the proximal; carpus longer than wide, longer than the propodus, with a cluster of 4 long setae on anterior margin, and 8 or 9 setae on posterior lobe; carpus wider than merus; propodus subrectangular, palm transverse with marginal and submarginal setae. Gnathopod 2, basis longer than wide; carpus triangular, much shorter than propodus, free portion of carpus posterior lobe approximately equal to width of merus; propodus palm oblique, regular, with no distinct notch or truncated process, posteroproximal corner produced into distinct rounded lobe with a few robust setae. Pereiopods (Fig. 8), 3 and 4 similar in size and shape, merus anterodistal lobes extending approximately 1/3 or less the length of the carpus; pereiopod 5 distinctly shorter than 6 and 7, which are similar in length; pereiopods 5-7 basis posterior margin expanded, more so in 5 and 7 than in 6; pereiopod 7 basis posterior lobe ventral margin without dentition or setae. Pleopods, long and slender, all with 1 to 2 coupling hooks on peduncle. Uropod 1 (Fig. 9) longer than uropod 2. Uropod 1, peduncle longer than rami, which are similar in length; uropod 2, rami similar in length; uropod 3 extending approximately as far as uropod 2 peduncle distal margin, ramus shorter than peduncle, ramus approximately as long as longest peduncle seta, ramus setae robust, as thick as peduncle setae. Telson, tapered to a rounded point, terminal setae closely set; terminal setae clearly finer than setae of uropod 3 peduncle and ramus. Allotype Female (Fig. 10). Similar in structure to male except for as follows. Gnathopod 2 similar in structure to gnathopod 1 (which is similar to gnathopod 1 of males),

820 JOURNAL OF CRUSTACEAN BIOLOGY, VOL. 35, NO. 6, 2015 Fig. 4. Hyalella spinicauda. INHS 14856, holotype male 5.76 mm. P, pereiopod. propodus longer than carpus, longer than propodus of gnathopod 1. Setose oostegites present on gnathopod 2 and pereiopods 3-5. Other Variations. Some individuals only have dorsal mucronations on pleonite 1. In rare individuals, pereiopod 7 basis posterior lobe may have one or two dentations with very fine setae. Etymology. wellborni; in compliment to Dr. Gary A. Wellborn for his contribution to the understanding of the evolution and ecology of this species complex.

SOUCEK ET AL.: TWO NEW SPECIES OF NORTH AMERICAN HYALELLA 821 Fig. 5. Hyalella spinicauda. INHS 14856, holotype male 5.76 mm. U, uropod; T, telson. Disposition of Types and Material Examined. The holotype and allotype (INHS 14859 and 14860) for H. wellborni were collected from Flat Branch, near Humboldt, IL (Table 1) and deposited in the INHS Crustacean Collection. Paratypes (INHS 14861) collected from Flat Branch (Table 1) were deposited in the INHS Crustacean Collection, and additional paratypes (YPM IZ 076755) collected from Panther Creek, Perry County, IL (Table 1), were deposited in the Yale University Peabody Museum of Natural History. Notes on Habitat and Range. Individuals of this new species were collected from both wadable lentic and lotic habitats in Illinois, Michigan, Indiana, and Texas, USA (Table 1), but more fieldwork is needed to determine its full range. The type material was collected from submerged macrophytes and cobble in a run in Flat Branch, near Humboldt, IL. Separation of H. azteca, H. spinicauda, and H. wellborni The two new species described here can be separated from H. azteca using characteristics of maxilla 1, gnathopod 2 of the males, the basis posterior lobe of pereiopod 7, and uropod 3 (Table 2; Figs. 11 and 12). (1) Hyalella azteca has at least 3 apical plumose setae on the inner plate of maxilla 1 (the lectotype has 3), whereas H. spinicauda and H. wellborni have 2 plumose setae. (2) In H. azteca the carpus of gnathopod 2 has a posterior lobe whose length is approx. 1.5 times the width of the merus of the same gnathopod, whereas in H. spinicauda and H. wellborni the length of this carpus posterior lobe is approximately equal to or shorter than the width of the merus (Fig. 11). (3) In H. azteca the propodus palm has a large rounded notch, whereas H. spinicauda has an angled step, and H. wellborni has neither (Fig. 11). (4) In H. azteca the distal margin of the posterior lobe of the basis of pereiopod 7 has at least two relatively long, robust spines, whereas in H. spinicauda and H. wellborni this posterior lobe has fine, short setae if any at all (Fig. 12). (5) In H. azteca the ramus of uropod 3 is much longer than the longest apical seta of the peduncle of uropod 3 (shown in Figure 2 of Gonzalez and Watling (2002)), whereas in H. spinicauda and H. wellborni, theramusof uropod 3 is only slightly longer than the longest apical seta of the peduncle uropod 3 (Figs. 5 and 9). In addition, based on body size and head capsule length (Table 2), H. azteca would be considered the large ecomorph of Wellborn (1995b), whereas H. spinicauda and H. wellborni would be considered the small ecomorph. Finally, Hyalella spinicauda and H. wellborni can be separated from each other using characteristics of gnathopod 2 of males, and the setation of uropod 3 and the telson (Table 2). (1) In H. spinicauda the palm of the gnathopod 2 propodus has an angled step (sometimes high magnification is required to see this), whereas in H. wellborni the gnathopod 2 propodus palm is regular, with no angles step or notch (Fig. 11). (2) In H. spinicauda the tip of the dactyl of the gnathopod 2 propodus approximately aligns vertically with end of the posterior lobe of the carpus, whereas in H. wellborni, the tip of the dactyl of gnathopod 2 propodus aligns vertically well beyond (posteriorly) the end of the posterior lobe of the

822 JOURNAL OF CRUSTACEAN BIOLOGY, VOL. 35, NO. 6, 2015 Fig. 6. Hyalella wellborni. INHS 14859, holotype male 4.28 mm. UL, upper lip; LL, lower lip; Mxpd, maxilliped; Mx1, maxilla 1; Mx2, maxilla 2; lmd, left mandible; rmd, right mandible. carpus (see dashed line, Fig. 11). Because this character may vary depending on limb flexion, we have not included it in the diagnosis or description. However, we retain it here and in Table 2 as an additional useful character for separation

SOUCEK ET AL.: TWO NEW SPECIES OF NORTH AMERICAN HYALELLA 823 Fig. 7. Hyalella wellborni. INHS 14859, holotype male 4.28 mm. Gn 1, gnathopod 1; Gn2, gnathopod 2. of the two species because the difference is great enough to make a determination in spite of limb flexion influence. (3) In H. spinicauda, the terminal setae of the telson are approximately equal in stoutness to the longest seta of the ramus of uropod 3, whereas in H. wellborni the terminal setae of the telson are much finer than the longest seta of the ramus of uropod 3 (Figs. 5 and 9). DISCUSSION The two new species described here are placed in the subgenus Hyalella Smith based on overall morphological similarity. Both species are easily distinguished from H. azteca (based on comparison to the lectotype and as redescribed by Gonzalez and Watling (2002)) as detailed above, but they are also distinct from previously described species that were later synonymized with H. azteca. We were not able to directly examine the type specimen of H. knickerbockeri designated by Bate (1862) as Allorchestes knickerbockeri and photos we obtained of the type did not provide sufficient detail to distinguish them, but the description by Bate (1862) of the first gnathopod states that the merus is broader than the carpus, and that the propodus is very long (this is also shown in a figure for the description). Both of the species described here have first gnathopods with a normal, i.e., not elongated, propodus, and the carpus is broader than the merus in both cases. Examination of a female syntype of H. dentata (Smith, 1874) (YPM IZ 009143) allowed separation from the two species described here using the relative lengths of the uropod 3 peduncle spines and the U3 ramus. In H. dentata the uropod 3 peduncle spines reach far short of half the length of the ramus, whereas in both of the new species described here, the peduncle spines nearly reach the end of the ramus (Figs. 5 and 8). The species described here can be distinguished from H. knickerbockeri because as figured in Weckel (1907), its carpus posterior lobe of gnathopod 2 is much longer than the width of the merus of the same gnathopod, and the ramus of uropod 3 is much longer than the longest seta of the peduncle of uropod 3. Weckel (1907) did not designate types as she was transferring the previously described Allorchestes knickerbockeri to the newly described erected genus. Lockingtonia fluvialis was vaguely described, with no figures. It could be any number of species of Hyalella. Hyalella faxoni is distinguished from the two new species here because it did not have poste-

824 JOURNAL OF CRUSTACEAN BIOLOGY, VOL. 35, NO. 6, 2015 Fig. 8. Hyalella wellborni. INHS 14859, holotype male, 4.28 mm. P, pereiopod. rior dorsal mucronations, and the maxilla 1 inner plate had 3 plumose setae. Hyalella ornata as figured (Pearse, 1911) is distinguished because of length of carpus posterior lobe on gnathopod 2, and the presence of three plumose setae on inner plate of maxilla 1. This species also had a large notch in palm of gnathopod 2 propodus similar to that of H. azteca,

SOUCEK ET AL.: TWO NEW SPECIES OF NORTH AMERICAN HYALELLA 825 Fig. 9. Hyalella wellborni. A, INHS 14859, holotype male, 4.28 mm (U1-U3). B, INHS 14861, paratype male, 4.01 mm (T). U, uropod; T, telson. unlike the two new species described here. Furthermore, the description of H. azteca in Bousfield (1996) is distinct from the species described here because the ramus of uropod 3 is much longer than the longest setae of the peduncle, although the inner plate of maxilla 1 is shown to have only two plumose setae. Gonzalez and Watling (2002) contend that while H. ornata is likely a synonym of H. azteca, H. faxoni, H. knickerbockeri, H. dentata, and H. inermis are all probably valid species. However, it is clear that the two species described here do not match any of these older descriptions. Weckel (1907) minimized the importance of the number of plumose setae on the inner plate of maxilla 1, but we feel this is an important taxonomic character. Having looked at large numbers of both new species described here from multiple collection sites, we never observed more than two plumose setae. However, laboratory cultured and field collected specimens that fit the Gonzalez and Watling (2002) description of H. azteca and match the characteristics of the H. azteca (Saussure, 1858) lectotype never had less than three plumose setae, although they sometimes had more than three (four or even five). Lazo- Wasem and Gable (1989) made this same observation for a population of H. azteca from Bermuda. In his key to the species of Hyalella, Baldinger (2004) separates H. azteca and H. texana from H. montezuma by the presence of an inner plate of maxilla 1 with one or two apical plumose setae, but the original type material of Saussure (1858) had three plumose setae (personal observation of lectotype, and see Fig. 2 of Gonzalez and Watling, 2002). Stevenson and Peden (1973) also showed three apical plumose setae in their figures of H. azteca for comparison to their description of H. texana. Given our observations of the consistency of this trait, we suggest that a specimen must have at least three apical plumose setae on the inner plate of maxilla 1 to be considered H. azteca. In the key to currently described species of Hyalella by Baldinger (2004), the first couplet separates species with dorsal mucronations from those without dorsal mucronations. While we have maintained this separation in our updated key, we feel this character may be problematic and requires further investigation. Wellborn et al. (2005) identified a population of small ecomorph Hyalella in Oklahoma that had only one dorsal mucronation (on pleon segment one). This population fit genetically within the C-clade of Wellborn and Broughton (2008) as do specimens of H. wellborni new species according to Major et al. (2013; Flat Branch clade). However, all of the specimens examined for this study that fit the description of H. wellborni had dorsal mucronation on both pleon segments one and two. In addition, specimens collected by Major et al. (2013) from New Mexico (population RC-NM) did not have any dorsal mucronations and keyed most closely to H. inermis, but genetically, they fell into what Major (2012) called the Burlington clade. All of the other members of that clade had dorsal mucronations. Using the 1569 bp COI data set, the population RC-NM specimens differed from other members of the Burlington clade by an average of 3.4% (K2P corrected distance; Major et al., 2013). Costa et al. (2009) found a

826 JOURNAL OF CRUSTACEAN BIOLOGY, VOL. 35, NO. 6, 2015 Fig. 10. A, Hyalella spinicauda. INHS 14857, allotype female, 4.82 mm. B, Hyalella wellborni. INHS 14860, allotype female, 5.04 mm. Gn, gnathopod.

SOUCEK ET AL.: TWO NEW SPECIES OF NORTH AMERICAN HYALELLA 827 Table 2. Morphological differences between H. azteca, H. spinicauda,andh. wellborni. a Head capsule length of adults at least two months old in laboratory culture. b As described by Wellborn (1995). c Illustrated in Fig. 11, could be influenced by limb flexion so caution is required. H. azteca H. spinicauda H. wellborni Head capsule length a >0.6 mm <0.5 mm <0.5 mm Morphotype b Large Small Small Maxilla 1 inner plate 3 pappose setae 2 pappose setae 2 pappose setae G2 ( ) carpus posterior lobe length Approx. 1.5 width of merus width of merus width of merus G2 ( ) propodus palm Large rounded notch Angled step No distinct step/notch G2 ( ) propodus dactyl vertical alignment c carpus post. lobe carpus post. lobe Far beyond carpus post. lobe P7 basis posterior lobe distal/bottom margin 2 stout spines Very short setae if any Very short setae if any U3 ramus length vs. longest seta on peduncle Much longer U3: thickness of longest seta of ramus Clearly finer or slightly less robust vs. setae of peduncle Telson terminal setae vs. longest seta Clearly finer of U3 ramus maximum conspecific divergence in the genus Gammarus of 4.30%, whereas Witt et al. (2006) used a screening threshold of 3.75% to distinguish provisional species of Hyalella. It is clear that further work should be done to better determine the extent to which presence (and number) of dorsal mucronations are diagnostic of different species of Hyalella. Numerous studies since the 1990s have suggested that Hyalella azteca is a species complex, and it is clear that morphological studies are lagging far behind genetic studies. Partial COI sequences have been generated for several described species including H. sandra, and H. muerta (Witt et al., 2006), H. montezuma (Witt et al., 2003), H. texana (Wellborn and Broughton, 2008), and H. azteca (Major et al., 2013; and several others). Complete COI sequences are available for the two new species described here as well. H. spinicauda was genetically identified as belonging to the Clear Pond clade by Major et al. (2013), and they provide names given to this clade by other researchers in their table 3. Major et al. (2013) identified a number of different haplotypes that fit within this clade, and complete cytochrome oxidase subunit 1 (COI) sequences for haplotypes from the type locality can be found under Genbank accession Nos JX446339 and JX446340. Hyalella wellborni was genetically identified as belonging to the Flat Branch clade by Major et al. (2013) (see their table 3 for other names given to this clade), and again a number of haplotypes of this clade were identified. Complete cytochrome oxidase subunit 1 (COI) sequences for the haplotypes from the type locality can be found under Genbank accession Nos JX446324, Fig. 11. Second gnathopods (propodus, carpus, and merus only) of (A) H. azteca,(b) H. spinicauda, and(c) H. wellborni. For each species, bars on carpus posterior lobe and merus are equal in length. Vertical dashed line shows relative position of dactyl and carpus posterior lobe. Arrows point the large rounded notch in the palm of H. azteca and the angled step in the palm of H. spinicauda.

828 JOURNAL OF CRUSTACEAN BIOLOGY, VOL. 35, NO. 6, 2015 Fig. 12. Seventh pereiopods (basis, ischium and merus only) of (A) Hyalella azteca, (B) H. spinicauda, and(c) H. wellborni. Arrows show presence or lack of spines on distal margin of basis posterior lobe. JX446325, and JX446326. In the present case, identification of morphological characters that separate the two species was achieved as a result of the fact that the populations were genetically identified. The use of genetic identity to inform morphological description may prove fruitful with a number of other species, particularly in the arid southwestern USA (Witt et al., 2006). KEY TO SPECIES OF Hyalella (Hyalella) IN NORTH AMERICA AND THE CARIBBEAN REGION (MODIFIED FROM BALDINGER, 2004, AND MARRÓN-BECERRA ET AL., 2014) 1 Eyesabsent...2 Pigmentedeyespresent...3 2 Antenna 1 longer than antenna 2; sternal gills on pereonites3-7...h. muerta Antenna 1 shorter than antenna 2; sternal gills on pereonites2-7...h. cenotensis 3 Body with dorsal mucronations..................... 8 Body without dorsal mucronations.................. 4 4 Ramus of uropod 3 vestigial or robust, subequal or shorter in length than peduncle..................... 5 Ramus of uropod 3 slender, subequal or longer in length than peduncle..................................... 6 5 Ramus of male uropod 3 robust with seven apical spines...h. sandra Ramus of male uropod 3 vestigial with two to four spines...h. meraspinosa 6 Antenna 1 and 2 subequal in length................. 7 Antenna 2 nearly twice the length of antenna 1...........H. longicornis 7 Hind margin of merus of pereopods 3 and 4 with long setae, telson with two closely set apical setae.............h. caribbeana or H. squamosa Hind margin of article 4 of pereopods 3 and 4 with short setae, telson with two long broadly spaced apical setae...h. inermis 8 Inner plate of maxilla 1 narrow, with two to five apical plumosesetae...9 Inner plate of maxilla 1 broad, subtriangular with two or three apical plumose setae followed closely by 22-30 similarmedialsetae...h. montezuma 9 Antenna 1 greater than half the length of antenna 2, only first, or first two abdominal segments bearing dorsal mucronations...10 Antenna 1 less than half the length of antenna 2, all three abdominal segments bearing dorsal mucronations.........h. texana 10 Gnathopod 2 of males, carpus posterior lobe about as long as width of merus; Uropod 3, ramus approximately as long as or slightly longer than longest seta on peduncle; Pereopod 7, distal/bottom margin of basis posterior lobe dentate or not dentate, with one or two verysmallsetaeifany...11 Gnathopod 2 of males, carpus posterior lobe 1.5 as long as width of merus; Uropod 3, ramus much longer than longest seta on peduncle; Pereopod 7, distal/bottom margin of basis posterior lobe strongly dentate and with two or more relatively long spines........... H. azteca 11 Gnathopod 2 propodus (males), palm without a distinct angled step, tip of dactyl aligning vertically well beyond (posteriorly) distal end of posterior lobe of carpus;

SOUCEK ET AL.: TWO NEW SPECIES OF NORTH AMERICAN HYALELLA 829 Telson terminal setae clearly finer than setae on U3 ramus...h. wellborni Gnathopod 2 propodus (males), palm with a distinct angled step (visible under high power), tip of dactyl approximately aligning vertically with distal end of posterior lobe of carpus; Telson terminal setae at least as stout as setae on U3 ramus........... H. spinicauda ACKNOWLEDGEMENTS Special thanks to M. J. Wetzel (INHS), E. R. DeWalt (INHS), and W. P. Norwood (Environment Canada) for providing specimens collected from locations outside of Illinois. Dr. Steven J. Taylor (INHS) assisted DJS with drawings of some characters. REFERENCES Baldinger, A. J. 2004. A new species of Hyalella (Crustacea: Amphipoda: Hyalellidae) from Ash Springs, Lincoln County, Nevada, USA, with a key to the species of the genus in North America and the Caribbean region. Journal of Natural History 38: 1087-1096., W. D. Shepard, and D. L. Threloff. 2000. 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