The Hyalella species flock of Lake Titicaca (Crustacea: Amphipoda): perspectives and drawbacks of dna-based identification

Lake Titicaca, in the High Andes of Perú and Bolivia, harbours the world’s third most speciose ancientlake amphipod radiation on record. A minimum of nineteen species of Hyalella derived from at least five independent colonization episodes concentrate in this high altitude water body, although the actual species number present has not yet been established and could be much higher. Herein, we take advantage of the description of three new species (H. krolli, H. gonzalezi, and H. hirsuta) and the re-description of other two (H. solida and H. nefrens) to assess the feasibility of adopting a dna-based identification approach to Contributions to Zoology (2021) 1-54


Introduction
At 3,810 m elevation in the Andean Altiplano of Perú and Bolivia, Lake Titicaca harbours one of the few cases of a large endemic species flock on record among lacustrine amphipod crustaceans. Only the Caspian "Sea" -placed at the southeast boundary between Europe and Asia -and Lake Baikal in southern Siberia, show higher levels of species richness, whereas other two ancient lakes, Lake Ohrid in the Balkans and Lake Fuxian in Junnan, China, show comparable numbers. All these lakes depart from the ordinary condition of continental waters, where amphipods do not reach the diversity and ecological disparity found in marine habitats.
In the Baikal (31.722 km2, 1.642 m maximum depth) -where no amphipods of direct marine derivation occur -there are around 354 species recorded thus far distributed among 39 genera and seven families (Takhteev, 2019). This remarkably high diversity represents about 5% of the grand total of freshwater amphipods known worldwide (Takhteev, 2000). Despite their broad morphological and ecological disparity (see below), molecular phylogenies suggest that all Baikalian taxa derive from Gammarus Fabricius, 1775 (Gammaridae) and appear nested within this genus (Hou & Sket, 2016).
Lake Ohrid (358 km2, 289 maximum depth), between Macedonia and Albania, harbours at least 17 taxa belonging to three primarily freshwater families: Crangonyctidae Bousfield, 1973(one species of Synurella Wrzesniowski, 1877, Niphargidae (five species of Niphargus) and Gammaridae (11 morpho-species of Gammarus). All these taxa except two of the Gammarus are endemic to the lake and its shore springs (Grabowski et al., 2017).
The Titicaca (8.300 km2, 281 m maximum depth) shows lower amphipod diversity than the aforementioned two larger lakes since it is geologically younger and occupies a considerably smaller surface. All taxa found here are unambiguously assignable to Hyalella S.I. Smith, 1874(Fam. Hyalellidae Bulycheva, 1957, which otherwise is the only genus of epigean amphipod known to occur in continental waters of South America. Furthermore, ecological disparity among these species has not proceeded as far as in the Caspian or the Baikal. No truly gigantic or dwarf forms, commensals of other invertebrates, brood parasites of other amphipods, permanent burrowers, bentho-pelagic migrants, or fully nektonic pelagic species similar to those found in the two aforementioned lakes, have been recorded thus far in the Titicaca (Barnard & Barnard, 1983;Takhteev, 2000).
The number of species present in the Titicaca has not been precisely established, although estimations by different specialists suggest it should not exceed of a hundred species (Bousfield, 1982;Hoffman, 2006;Crawford et al., 1993). Nineteen species have been recorded thus far, of which 15 are apparently endemic (Faxon, 1876;Chevreux, 1904Chevreux, , 1907Weckel, 1910;González & Watling, 2001González & Coleman, 2002;Coleman & González, 2006). This number represents about one-quarter of the total Hyalella found in the Neotropics, where about 74 species are known to occur.
The Hyalella of the Titicaca are remarkable in their morphological disparity, largely due to the presence of species with a strongly armoured body integument. These "processiferous" forms -with spines, keels and/or flanges developed on body tergites and pereiopodal coxal plates -recall the aspect of members of some typical marine families such as the Epimeriidae Boeck, 1871, Iphimediidae Boeck, 1871, Dexaminidae Leach, 1814 or Atylidae Lilljeborg, 1865, but are rarely reported among epigean freshwater amphipods. Only some Baikal species (Takhteev, 2000(Takhteev, , 2019, some Caspian Gammaroids (Sars, 1894(Sars, , 1896 and oddities such as Fuxiana yangi Sket, 2000, from Lake Fuxian (China;Sket, 2000) and Issykogammarus hamatus Chevreux, 1908, from Lake Issyk-Kul (Kazakhstan;Chevreux, 1908) display such a comparable armoured condition. In the case of the species of the Titicaca, the development of this armature might be related to the predation pressure exerted by the Cyprinodontid killifish endemic to the Lake (Orestias Valenciennes, 1839; up to 24 spp.; Lauzanne, 1992), but this is still not firmly established (González & Coleman, 2002).
Recently, Adamowicz et al. (2018) showed based on molecular markers that the Hyalella of the Titicaca are not monophyletic but derive from at least five independent colonization episodes. Furthermore, Jurado-Rivera et al. (2020) and Zapelloni et al. (2021) disclosed new hyalella (CRUSTACEA, AMPHIPODA) from lake titicaca | 10.1163/18759866-bja10021 a high discordance between morpho-species and Molecular Operational Taxonomic Units (motus) within this assemblage. The derivation from multiple colonization events and the incongruence between morpho-species and motus seem to be a hallmark of the species radiations arisen in ancient lakes and oceanic islands (Monaghan et al., 2006;Critescu et al., 2010).
It is high time to address the true magnitude of the Titicacan Hyalella species diversity. Increasing levels of eutrophication due to urban sewage discharge in populated bays, and the industrial pollution derived from mining across the lake watershed, pose significant threats to the conservation of this remarkable species assemblage (Archundia et al., 2017). An even greater menace is represented by two predatory fish species introduced into the lake: Onkorhynchus mikiss (Walbaum, 1792) and Odontesthes bonariensis (Valenciennes, 1835), with the former being extensively cultured in net-cages (Vila et al., 2007).
Here we contribute to the revision of the Hyalella amphipods of Lake Titicaca introducing the description of three new species while re-describing other two. One of the new taxa corresponds to a shallow water form with a smooth, compact body, shortened antennules and antennae, and strongly spinose, shortened posterior pereiopods that remind the condition exhibited in some typically fossorial marine forms such as the Haustoriidae Stebbing, 1906, or the Phoxocephalidae G.O. Sars, 1891. This new taxon is remarkable also in the display of hardly sexually dimorphic second gnathopods. The second new species is a deeper water form that also shows an unarmoured body but displays a transverse dorsal hump along the posterior margin of body tergites. The third new species corresponds again to a smooth body, deep-water form that seems to be limited to occur on unconsolidated murky sediments. It is characterised by the display of numerous long setae along the medial margin of anterior pereiopods, a feature reminiscent to the condition of some limnic Gammaridae. Furthermore, we redescribe H. solida Chevreux, 1907, a species characterised by displaying a hispid integument and the presence of five dorsal flanges; it was originally described based on a single specimen and had not been reported again since its discovery. Finally, we redescribe H. nefrens González & Watling, 2003, a species with a peculiar, untoothed mandible and three pointed dorsal processes, emphasising the variable expression of the latter feature, otherwise profusely used in Hyalella taxonomy.
We have obtained dna sequences of the mitochondrially-encoded cytochrome c oxidase subunit I (= cox1) of four out of the five aforementioned species in addition to morphological descriptions. We take advantage of these data and of all previous molecular information gathered on the Titicacan Hyalella (Adamowicz et al., 2018;Jurado-Rivera et al., 2020) to explore the feasibility of adopting an integrative approach -combining morphological and molecular information -to unveil the actual species diversity present in the lake.

Material and methods
Specimens were collected directly with a hand-held plankton net or with a small dredge thrown either from the shore of the lake or from a boat and preserved in the field in 96% ethanol immediately after collection. Specimens were treated in the laboratory with lactic acid to remove internal tissues to facilitate observation. Drawings were prepared using a camera lucida on a Leica dm 2500 microscope equipped with Nomarski differential interference contrast. Body measurements were derived from the sum of the maximum dorsal dimensions of head, pereionites, pleonites and urosomites, including telescoped portions, and exclude telson length. Type material is deposited in the invertebrate collection of Naturalis Biodiversity Center, Leiden [rmnh].
Cox1 sequences and molecular phylogenetic analyses were obtained and performed following the protocols described in our previous study on the Hyalella from the Titicaca (Jurado-Rivera et al., 2020). The new dna sequences introduced herein were merged with the large dataset used in the aforementioned study and are freely available via genbank (see corresponding accession numbers in the corresponding sections on material examined below). Diagnosis.Body smooth, compact, with somites comparatively much broader than long. Eye ellipsoid. Antennules, antennae and pereiopods v-vii strongly shortened. Five pairs of sternal gills, one on each of pereionites iii-vii. Mandible incisor toothed, left 5-denticulate, right 6-denticulate. Maxillule (= Maxilla 1) basal endite (= outer plate) with nine rake-like spines; endopod (= palp) comparatively elongate (i.e., surpassing midway of distance between base of segment and distolateral angle of basal endite), with single serrulate seta on tip; seta as long as half segment length. Maxilla (= Maxilla 2) coxal endite (= inner plate) with single hipertrophied pappose seta proximally on medial margin. Male gnathopod ii propodus hardly sexually-dimorphic, slender, subrectangular, hardly expanded, about 1.5 times longer than broad, with palm margin much shorter than posterior margin. Coxal plate iv comparatively slender, much longer (1.5 ×) than broad. Pereiopods v-vii bristled with spines. Male uropod I endopod lacking modified curved spine. Uropod iii ramus much shorter than protopod. Telson rimmed with numerous (up to 10) slender setae, but devoid of spines.

Taxonomy
Etymology. Species name refers to the German malacologist Oliver Kroll, who collected all the material of the new taxon known.
length as: 1: 0.63: 0.40; main flagellum shortened (shorter than two distal segments of peduncle combined); accessory flagellum absent; three penultimate articles of main flagellum with one or two short simple aesthetascs.
Both mandibles with toothed incisor and triturative columnar molar; molar seta well developed on both; palp lacking. Left mandible ( fig. 2A) incisor and lacinia both 5-denticulate ( fig. 2B), latter with patch of long setules proximally; mandible setal row comprising three main setae and three accessory setae disposed in two parallel rows, all setae pappose; molar with patch of slender setae at base on distal margin as figured.
Maxilla ( Gnathopod I ( fig. 3A-C) subchelate with carpus 1.3 times longer than propodus, latter about 1.5 times longer than broad, with convex palm margin; palm angle marked with two unequal flagellate spines; up to seven bipectinate setae present on medial surface of propodus as figured.
Gnathopod ii ( fig. 4A, B) subchelate with carpus also longer than propodus, but latter comparatively more elongate than gnathopod I counterpart, about 1.7 times longer than broad.
Pereiopods v-vii (fig. 6A-C) strongly spinose, especially pereiopod V, resembling in some way condition exhibited by fosorial marine amphipods such as some members of the Haustoriidae or the Phoxocephalidae. Distal portion of pereiopods comparatively short (i.e., each with basis longer than corresponding ischium-merus combined). Basis of periopod vii broadest, only 1.2 times longer than broad; basis of pereiopods v-vi more slender, each 1.4 times longer than broad.
Uropod I (fig. 3F) both rami much shorter than protopod; latter with 3-4 spines along posterolateral margin and one spine on each posterolateral and posteromedial distal angle of segment. Exopod with one spine along posterolateral margin; endopod with two spines along posteromedial margin.
Uropod ii ( fig. 4C) shortened, total length not surpassing length of protopod of uropod I. Rami slightly shorter than protopod; latter with two spines along posterolateral margin and single spine on each posterolateral and posteromedial distal angle of segment; posteromedial margin naked. Exopod with four unequal spines distally. Endopod with five unequal spines distally and single slender spine about midway on posteromedial margin.
Uropod iii ( fig. 4D) with ramus much shorter than protopod; latter with three spines on distolateral angle and simple seta on distomedial angle; three more slender simple setae placed on medial surface of segment as figured. Ramus with spine and pair of simple setae on tip; spine shorter than half ramus length.
Telson ( fig. 4E) subrectangular, slightly broader than long, with about 10 simple setae along distal margin, median pair of setae longer than rest; integument densely microspinulose as figured.
Male. Body 9.59 mm long. As female in all respects except for presence of penile papillae, up to ten bipectinate setae (vs. up to seven in female) on medial surface of propodus of gnathopod I ( fig. 3G), and propodus of gnathopod ii. Latter ( fig. 4F) slender, 1.4 times longer than carpus, subrectangular, hardly expanded, about 1.5 times longer than broad, with palm margin much shorter than posterior margin; palm angle marked with two flagellate spines; medial surface of segment with transverse row of five bipectinate setae.
Remarks. This species is easily distinguished from the rest of representatives of the genus based on its compact aspect with shortened antennules and antennae, and shortened spiky pereiopods v-vii. As stated above, its aspect resembles in some way the condition exhibited in fossorial marine amphipods such as some members of the Haustoriidae or the Phoxocephalidae, but we are uncertain as regard the life habits of the new species. In any event, the two known localities where the species was collected correspond to shallow waters (up to 1.5 m depth) covered with macrophytes.
The hardly developed sexual dimorphism on male gnathopod ii is also a remarkable feature of the new taxon, not found in any other Hyalella species, and reminds the condition exhibited in some talitrids (e.g., Talitrus Latreille, 1802).
As it will be stated below, this species arises in molecular phylogenetic analyses together with H. gonzalezi Jaume sp. nov. -described below -as the closest relative of Hyalella armata, perhaps the most bizarre of all Titicacan species, characterised by the display of an extremely elongate, laterally-directed spine on each pereiopodal coxal plate i-iv (see González & Coleman, 2002). Whereas in H. gonzalezi Jaume sp. nov. the close kinship with H. armata (Faxon, 1876) could be at the base of the common display of peculiar morphological features such as the presence of body dorsal humps, the truncate endopod (= palp) of maxillule with a comparatively long distal seta, the male gnathopod ii propodus of triangular outline and with the two spines on palm angle comparatively elongate, and a third uropod with shortened ramus, there is nothing in the morphology of H. krolli Jaume sp. nov. denoting its closeness to H. armata except perhaps the truncate endopod of maxillule with a comparatively long distal seta and the short ramus of uropod iii.  Diagnosis. Body smooth, with faint but distinct transverse hump along posterodorsal margin of pereionites and pleonites; body integument strongly tinged dark when alive. Eye circular. Five pairs of sternal gills, one on each of pereionites iii-vii. Incisor of both mandibles toothed, 6-cuspidate. Maxillule (= Maxilla 1) basal endite (= outer plate) with nine rake-like spines, three of them spatulate; endopod (= palp) comparatively elongate (i.e., surpassing midway of distance between base of segment and distolateral angle of basal endite), with single serrulate distal seta; seta comparatively elongate, about as long as segment. Maxilla (= Maxilla 2) coxal endite (= inner plate) with distal margin lined with series of modified slender spines provided with series of transverse lamellae; only one hypertrophied pappose seta present proximally on medial margin of endite. Male gnathopod ii propodus massive, triangular; palm margin as long as posterior margin. Coxal plate iv comparatively slender, much longer (1.4 ×) than broad, with deeply excavate posterior margin. Male uropod I endopod lacking modified curved spine. Uropod iii ramus much shorter than protopod. Telson devoid of spines, with about five simple setae along distal margin.
Etymology. Species name refers to the Chilean Hyalella specialist Prof. Exequiel González Balbontín, one of the main contributors to the study of the species of Lake Titicaca.
Both mandibles with toothed, 6-cuspidate incisor and triturative columnar molar; latter with patch of short slender pappose setae and long setules at base of anterior margin as figured; molar seta well developed on both branches; palp lacking. Left mandible ( fig. 8A, B) lacinia 5-cuspidate with proximal portion of inner surface covered with densely set short spinules. Mandible setal row comprising three main pappose setae and three accessory pappose setae disposed in two parallel rows (3 + 3).
Right mandible ( fig. 9C, D) lacinia trifid, with series of round marginal denticles on each cusp distributed as figured, and with patch of densely set short spinules on proximal portion of inner surface. Mandible setal row comprising 2 + 2 pappose setae.
Maxilla ( Pereiopods iii-iv ( fig. 11A, B) about similar in length, ordinary; both with posterior margin of carpus and propodus lined with spines.
Pereiopods v-vii ( fig. 12A-C) elongate, each with basis about as long as corresponding ischium-merus combined. Basis of pereiopod vi not as strongly expanded posteriorly nor with posterodistal angle so strongly overhanging as in pereiopods V and vii, with straight posterior margin.
Uropod I ( fig. 11C) rami shorter than protopod, latter armed with four spines along posterolateral margin and one spine on each posterolateral and posteromedial distal angle of segment. Exopod with one spine midway on posterolateral margin. Endopod lacking modified distal spine, with two spines along posteromedial margin.
Uropod ii ( fig. 8E) rami shorter than protopod, latter armed with three spines along posterolateral margin and one spine on each posterolateral and posteromedial distal angle. Exopod with spine midway on posterolateral margin. Endopod with spine on posteromedial margin.
Uropod iii ( fig. 8F) with ramus much shorter than protopod, latter with three short spines on distolateral angle and three simple setae along medial margin. Ramus with short spine and three simple setae on tip; spine less than half ramus length.
Telson (Fig. 8G) broader than long, with evenly rounded posterior margin. Armature consisting on about five slender simple setae along distal margin and three short penicillate setae subdistally at each side.
Brooding female. Body up to 10.7 mm long. As male in all respects except for presence of oöstegites on gnathopod ii and pereiopods iii-v, and structure of gnathopod ii. Latter ( fig. 10C) with propodus slender, slightly longer (1.1 ×) than carpus and 1.7 times longer than broad; palm angle marked with two flagellate spines; palm margin slightly transverse, convex, armed as in fig. 10D; medial surface of segment with row of six bipectinate spines as figured.
Remarks. This new species is remarkable for the display of a faint but distinct transverse hump along the posterior margin of tergites of pereionites and pleonites, in an otherwise smooth body. Furthermore, it displays a peculiar ornamentation composed of transverse lamellae on the spines of one of the series present on the coxal endite (= inner plate) of maxilla. Similar body dorsal humps occur in other two species, both also endemic to Lake Titicaca: H. armata (Faxon, 1876) and its closely related sibling H. longispina González & Coleman, 2002. The latter two species share the display of a long transverse spine on each of pereiopodal coxal plates i-iv, conferring them one of the more peculiar aspects among the Titicacan species, and readily tell them apart from the new species. As previously stated above in the remarks on H. krolli Jaume sp. nov., the latter species and H. gonzalezi Jaume sp. nov. arise as the closest relatives of H. armata in molecular phylogenetic analyses. The last two species share the display of body dorsal humps, a truncate endopod (= palp) of maxillule with a comparatively long distal seta, a male gnathopod ii propodus of triangular outline and with the two spines on palm angle comparatively elongate, and a third uropod with shortened ramus.
With regard the row of spines provided with transverse lamellae present on the coxal endite (= inner lobe) of maxilla, similar structures have been reported to occur only in H. nefrens (see redescription below) aside the new species.
The gut contents of one of the specimens analysed included remains of a large anomopod branchiopod crustacean (Simocephalus sp.).   Diagnosis. Body smooth. Eye ellipsoid. Five pairs of sternal gills, on pereionites iii-vii. Incisor of both mandibles toothed, left 6-cuspidate, right 5-cuspidate. Maxillule (= Maxilla 1) basal endite (= outer plate) with nine rakelike spines; endopod (= palp) short (i.e., much shorter than half of distance between base of segment and distolateral angle of basal endite), with single simple distal seta; seta reduced, less than half length of segment. Maxilla (= Maxilla 2) with only one hypertrophied pappose seta proximally on medial margin of coxal endite (= inner plate). Male gnathopod ii propodus massive, triangular; palm margin as long as posterior margin. Coxal plate iv as long as broad, with deeply excavate posterior margin. Pereiopods iii-v hairy. Male uropod I endopod with short, faintly modified subterminal spine. Uropod iii ramus shorter than protopod. Telson about as long as broad, devoid of spines, with one pair of unequal simple setae at each side on distal margin.

Hyalella hirsuta
Etymology. From Latin hirsutus (= hairy), species name refers to the series of long simple setae running along the medial margin of merus and carpus of pereiopods iii-v.
Distribution. Endemic to Lake Titicaca. Male. Body (fig. 13A) up to 9.0 mm long, smooth. Eye ellipsoid. Head about equal in length to pereionites i-ii combined; head lobe evenly rounded. Five pairs of sternal gills ( fig. 17C), one on each of pereionites iii-vii. Epimeral plates unarmed; distal margin of plate I evenly rounded; plates ii-iii posterodistally acuminate but with angles hardly produced.
Right mandible ( fig. 18A) with 5-cuspidate incisor and trifid lacinia, latter with series of round marginal denticles on each cusp distributed as figured, and with patch of densely set short spinules on proximal portion of inner surface. Mandible setal row comprising 2 + 2 pappose setae.
Maxilla ( Gnathopod I ( fig. 16D-F) subchelate; propodus much shorter than carpus (attaining about 82% length of carpus), ~1.4 times longer than broad, expanded distally; up to eight bipectinate setae in a row on medial surface of segment as figured; palm angle marked by two unequal flagellate spines; palm margin convex, armed as figured.
Gnathopod ii ( fig. 17A) subchelate; propodus massive, triangular, 1.2 times as long as broad with posterior margin about as long as palm margin; palm angle placed at about 43% length of segment, marked with two reduced flagellate spines; palm margin strongly armoured ( fig. 17B).
Pereiopods iii-iv ( fig. 18B, C) about similar in length; both with medial margin of merus and carpus lined with numerous long simple setae.
Pereiopods v-vii ( fig. 19A-C) elongate, each with basis about as long as corresponding ischium-merus combined. Basis of pereiopod vi not as strongly expanded posteriorly nor with posterodistal angle so strongly overhanging as in pereiopods V and vii, with straight posterior margin. Pereiopod V strongly setose, especially on merus ( fig. 19A).
Uropod I ( fig. 18D) rami shorter than protopod, latter armed with three spines along posterolateral margin, one spine on posterolateral distal angle, and single slender spine placed subdistally on posteromedial margin. Exopod with two spines on posterolateral margin and four on tip. Endopod, with two spines along posteromedial margin and six disposed distally, of which one ( fig. 18E) slightly sexually dimorphic.
Uropod ii ( fig. 16G) rami longer than protopod, latter armed with three spines along posterolateral margin, one spine on posterolateral distal angle, and single slender spine placed subdistally on posteromedial margin. Exopod with two spines on posterolateral margin and four on tip. Endopod with two spines on posteromedial margin and six on tip.
Uropod iii ( fig. 15C) with ramus shorter than protopod. Latter with three spines on distolateral angle and two simple setae along medial margin; longest spine on distolateral angle as long as ramus. Ramus with short spine and three simple setae on tip; spine less than half ramus length.
Telson ( fig. 15D) subrectangular, about 1.3 times broader than long; distal margin with pair of unequal simple setae at each side.
Brooding female. Body up to 7.8 mm long. As male in all respects except for presence of oöstegites on gnathopod ii and pereiopods iii-v, and structure of gnathopod ii. Latter (Fig. 17D, E) with propodus slender, shorter (0.9 ×) than carpus and 1.8 times longer than broad, expanded distally; palm angle marked with two flagellate spines; palm margin slightly transverse, convex, armed as in fig.  17E; row of three bipectinate setae on medial surface of segment as figured.
Remarks. This species is characterized by the display of hairy pereiopods iii-v, resembling the condition exhibited in some continental water Gammaridae (v. Pinkster, 1993); no other Hyalella species shows pereiopods ornamented in such a way. Furthermore, the new species displays a faintly modified subterminal spine on the endopod of male uropod I; whereas most Hyalella species show either a conspicuously long and recurved modified spine or an ordinary spine at such placement, only H. cajasi Alonso & Jaume, 2017 from the highlands of southern Ecuador displays a spine faintly modified in the same way as the new species described herein (Alonso & Jaume, 2017).
The new taxon seems to favour relatively deep-water (up to 30 m depth), murky conchiferous bottoms mostly devoid of vegetation. Chevreux, 1907 (figs 13B, 20-25) Material examined. Perú with nine rake-like spines; endopod (= palp) short (i.e., much shorter than half of distance between base of segment and distolateral angle of basal endite), with single reduced (less than half length of segment) spine distally, Maxilla (= Maxilla 2) with one hypertrophied pappose seta proximally on medial margin of coxal endite (= inner plate). Male gnathopod ii propodus massive, triangular; palm margin as long as posterior margin. Coxal plate iv 1.3 times longer than broad. Male uropod I endopod without modified subterminal spine. Uropod iii ramus shorter than protopod. Telson broader than long, devoid of spines, with one long simple seta at each side on distal margin.
Left mandible ( fig. 21A) with toothed, 6-cuspidate incisor and 5-cuspidate lacinia, latter with patch of densely set short spinules on proximal portion of inner surface. Mandible setal row comprising two main pappose setae figure 20 Hyalella solida Chevreux, 1907, male Gnathopod I ( fig. 22B, C) subchelate; propodus slightly shorter than carpus (attaining about 93% length of carpus), ~1.7 times longer than broad, expanded distally; up to six bipectinate setae in a row present on medial surface of segment as figured; palm angle marked by two unequal flagellate spines; palm margin slightly convex, armed as figured.
Gnathopod ii ( fig. 23A, B) subchelate; propodus massive, triangular, 1.3 times as long as broad with posterior margin slightly shorter than palm margin; palm angle placed midway of segment, marked with seven short flagellate spines; palm margin armoured as in fig. 23B.
Uropod I ( fig. 25B) with rami shorter than protopod, latter armed with row of six spines along posterolateral margin, two spines along posteromedial margin, and one spine on each distolateral and distomedial angle of segment.
Exopod with row of four spines along posterolateral margin and four spines on tip; endopod with two spines on posterolateral margin, row of four along posteromedial margin, and six spines terminally; endopod lacking modified spine.
Uropod ii ( fig. 25C) rami about as long as protopod, latter armed with row of four spines along posterolateral margin, one reduced simple seta on posteromedial margin, and one spine on each posterolateral and posteromedial angle of segment. Exopod with three spines along posterolateral margin and four placed distally. Endopod with three spines along posteromedial margin and six disposed distally.
Uropod iii ( fig. 25D) with ramus shorter than protopod, latter with transverse row of six spines on distolateral angle and about six simple setae on medial margin. Ramus with short spine and cluster of about seven simple setae on tip; spine much shorter than half ramus length.
Telson ( fig. 25E) broader than long, with evenly rounded posterior margin and slightly concave lateral margins. Armature consisting on one simple seta at each side on distal margin and three short penicillate setae placed subdistally at each side.
Brooding female. Body up to 9.8 mm long. As male in all respects except for presence of oöstegites on gnathopod ii and pereiopods iii-v, and structure of gnathopod ii. Latter ( fig. 23C) with propodus slender, slightly shorter than carpus and 1.8 times longer than broad, expanded distally; palm angle marked with two long flagellate spines; palm margin slightly transverse, convex; three bipectinate setae on medial surface of segment as figured.
Remarks. This species is readily identified by its densely setulose body integument combined with the display of five pointed dorsal flanges positioned one on each of pereionites v-vii and pleonites i-ii. The strongly armoured uropods i-ii are also a remarkable feature of this taxon. It was described by Chevreux (1907) based on a single male specimen collected by the expedition of de Créqui Montfort and Sénéchal de La grande to the Titicaca and never found again since its discovery, to the point that González & Watling (2003) considered it was not possible to differentiate this species and even suggested that the setulation of body somites described by Chevreux (1907) might correspond to a case of infestation with protozoans or fungi.   Diagnosis. Body usually with three dorsal flanges, one on each pereionite vii and pleonites i-ii, rarely with two, one or none. Eye rounded. Five pairs of sternal gills, one on each pereionites iii-vii. Incisor of both mandibles smooth. Maxillule (= Maxilla 1) basal endite (= outer plate) with nine spines, of which three spatulate and hardly denticulated, rest rake-like; endopod (= palp) short (i.e., much shorter than half of distance between base of segment and distolateral angle of basal endite), unarmed. Maxilla (= Maxilla 2) with only one hypertrophied pappose seta proximally on medial margin of coxal endite (= inner plate); distal margin lined with series of modified slender spines provided with transverse lamellae. Male gnathopod ii propodus massive, triangular; palm margin as long as posterior margin. Coxal plate iv (1.3 times as long as broad, with deeply excavate posterior margin. Male uropod I endopod without modified subterminal spine. Uropod iii ramus about as long as protopod. Telson much longer than broad, with one short stout spine at each side on distal margin.
Male. Body (fig. 26) up to 12.3 mm long, usually with three pointed dorsal flanges, one on each of pereionite vii and pleonites i-ii, but occasionally with less or even none (see fig. 27). Eye rounded. Head about as long as pereionites i-ii combined; head lobe roughly triangular. Five pairs of sternal gills, one on each of pereionites iii-vii. Epimeral plates (figs 26-27) unarmed; plates ii-iii posterodistally acuminate.
Antenna (= Antenna ii) (figs 26, 32B) about as long as half body length, peduncle much longer than head; relative length of peduncle segments 4-5 as 0.73: 1. Flagellum as long as peduncle.
Left mandible ( fig. 28A) with smooth, untoothed incisor and lacinia, latter with patch of densely set short spinules on proximal portion of inner surface. Mandible setal row comprising three main pappose setae and three accessory pappose setae disposed in two parallel rows (3 + 3). Rest of mandible as figured.
Right mandible ( fig. 33A) with smooth incisor and sharp trifid lacinia, latter provided with sharp marginal denticles on each cusp distributed as figured, and with patch of densely set short spinules on proximal portion of inner surface. Mandible setal row consisting of 2 + 2 pappose setae.
Maxilla ( Gnathopod I ( fig. 30A-C) subchelate; propodus much shorter than carpus (attaining about 81% length of carpus), ~1.7 times longer than broad, expanded distally; up to nine bipectinate setae in a row on medial surface of segment as figured; palm angle marked by two unequal flagellate spines; palm margin strongly convex ( fig. 30C), armed as figured.
Gnathopod ii ( fig. 31A-C) subchelate; propodus massive, triangular, 1.3 times as long as broad with posterior margin slightly shorter than palm margin; palm angle protruding as rounded bulge, placed at 38% length of segment, marked with two reduced flagellate spines; palm margin armoured as in fig. 31C.
Pereiopods v-vii elongate ( fig. 33B-D), with basis much longer, shorter or equal to corresponding ischium-merus combined, respectively; basis of pereiopod vi with posterodistal angle not so strongly overhanging as in pereiopods V and vii.
Uropod I ( fig. 31D) with rami shorter than protopod, latter armed with row of six spines along posterolateral margin and single spine subdistally on each posterolateral and posteromedial angle of segment. Exopod with row of five spines along posterolateral margin and four spines on tip; endopod with five spines along posteromedial margin, subterminal row of up to five short spines (number variable, some specimens even with only one spine; fig.  31E) on posterolateral margin, and five spines on tip; endopod lacking modified spine.
Uropod ii ( fig. 29D) rami about as long as protopod, latter armed with row of five spines along posterolateral margin and one spine subdistally on each posterolateral and posteromedial angle of segment. Exopod with three spines along posterolateral margin and seven placed distally. Endopod with three spines along posteromedial margin and four disposed distally.
Uropod iii ( fig. 32E) with ramus about as long as protopod, latter with two short spines (exceptionally three or four) on distolateral angle and about three simple setae on medial margin. Ramus with short spine and cluster of about four simple setae on tip; spine much shorter than half ramus length.
Telson ( fig. 32F) much longer than broad, with evenly rounded posterior margin and straight lateral margins. Armature consisting on one flagellate spine at each side on distal margin and three short penicillate setae placed subdistally at each side.
Brooding female. Body up to 10.2 mm long. As male in all respects except for presence of oöstegites on gnathopod ii and pereiopods iii-v, and structure of gnathopod ii. Latter ( fig. 30D) with propodus elongate, slightly longer (~1.1 ×) than carpus and 2.4 times longer than broad, expanded distally; palm angle marked with two long flagellate spines; palm margin slightly transverse, convex; up to nine bipectinate setae in a row on medial surface of segment as figured.
Remarks. This species, described by González & Watling (2003), is remarkable for its smooth, untoothed mandibular incisors; no other Hyalella shows such a feature, otherwise rarely reported to occur among continental water amphipods (e.g., Jaume & Wagner, 1998), but quite frequent in marine forms. The species is notorious also for the display of three dorsal processes, one on each pereionite vii and pleonites i-ii, but availability of a large number of specimens for inspection has enabled here to fix the level of variability in the expression of both the number and development of these processes, which can vary from the usual three to display none ( fig.  27). Other remarkable features of this taxon include the much longer than broad telson, the unarmed endopod of maxillule and the row of lamellate spines of the coxal endite of maxilla, the last feature shared only with H. gonzalezi Jaume sp. nov., described above.

Integrative taxonomy
In a previous paper (Jurado-Rivera et al., 2020), we explored the Hyalella species diversity of South America based on mitochondrial cox1 sequences and implementing different molecular species delimitation criteria. We recorded the presence of at least eleven different Molecular Operational Taxonomic Units (motus) in the Titicaca, and the occurrence of a remarkable disagreement between morphology and genetic data within this assemblage. We reported cases in which several motus corresponded to the same morpho-species, while in others a particular motu was shared by a heterogeneous array of species, including some with smooth and others with heavily armoured body.
Our ongoing research on the morpho-species of the Titicaca also suggests that they cannot be diagnosed as straightforward as we tentatively proposed in a previous work (see dichotomic key to species in Jurado-Rivera et al., 2020), and that detailed redescriptions and sequence assignments are required to disclose the true magnitude of this species flock. Herein we formally describe two new species whose cox1 sequences were already used in our previous study, whereas describe and provide cox1 sequences of a third one not reported yet. Unfortunately, we failed in obtaining valid cox1 sequences of H. solida, but provide three new sequences of H. nefrens. All these data were merged in our Hyalella cox1 sequence dataset and analysed in the same way as described in Jurado-Rivera et al. (2020). Results ( fig. 34) confirm the non-resolutive power of adopting a barcoding approach to distinguish among these morphologically highly divergent species: whereas H. hirsuta Jaume sp. nov. and H. nefrens share the same motu (= A1), H. gonzalezi Jaume sp. nov. and H. krolli Jaume sp. nov. arise in molecular phylogenetic analyses as the closest relatives and sharing the same motu (= C1) as H. armata, perhaps the most unusual of all Titicacan species, characterised by the display of an extremely elongate, laterally-directed spine on each of pereiopodal coxal plates i-iv (González & Coleman, 2002). Whereas in H. gonzalezi Jaume sp. nov. the close kinship with H. armata could explain the common display of peculiar morphological features such as the presence of body dorsal humps and the truncate endopod (= palp) of maxillule with a comparatively long distal seta, there is nothing in the morphology of H. krolli Jaume sp. nov. denoting its closeness to H. armata except for the truncate endopod of maxillule displaying a comparatively long distal seta.
The morphological diversification and the incongruence between morphological and molecular boundaries as found in the Hyalella of the Titicaca could be related to new hyalella (CRUSTACEA, AMPHIPODA) from lake titicaca | 10.1163/18759866-bja10021 the sharp changes in hydrological conditions experienced by the lake in the past, which included dramatic fluctuations in water level and salinity (see Jurado-Rivera et al., 2020, and references herein). Such environmental shifts could have triggered rapid changes in the morphology and the ecological differentiation of the species, followed by phenotypic convergence among the diverse lineages. But the intervention of other factors such as phenotypic plasticity, incomplete lineage sorting, or admixture between divergent lineages, cannot be ruled out to explain the morphological-genetic incongruence described herein, since similar patterns have been recorded also among the Gammarus species flock of Lake Ohrid, which had a very stable geological history compared to the Titicaca (Wysocka et al., 2013).