Phylogenetics of Teleogramma, a Riverine Clade of African Cichlid Fishes, with a Description of the Deepwater Molluskivore—Teleogramma obamaorum— from the Lower Reaches of the Middle Congo River

ABSTRACT The lower Congo River and nearby habitats harbor numerous endemic lineages of cichlid fishes, including some with highly specialized morphologies. Based on morphological and molecular data, we herein describe a new species of Teleogramma, a member of the chromidotilapiine clade found on rocky outcrops in the lower reaches of the middle Congo River. The new species, T. obamaorum, is distinguished from congeners by numerous morphological and ecological attributes, including the lack of dorsoventral head and body depression, absence of sexual dichromatism, and features of laterosensory anatomy, pharyngeal and gut morphology, and dietary preference. Phylogenetic analyses of two nuclear and two mitochondrial loci using Bayesian and maximum-likelihood inference lend strong support for the taxonomic validity of T. obamaorum and provide preliminary estimates of species relationships within the genus. The discovery of a new, ecomorphologically distinctive cichlid species in the Congo River suggests that additional research focus on riverine clades has the potential to greatly contribute to our understanding of evolutionary dynamics in this hyperdiverse group of teleost fishes.


INTRODUCTION
Teleogramma is a small clade of rheophilic, rock-dwelling cichlids restricted to fast-flowing waters in the western Congo basin. Unlike members of most riverine cichlid lineages, which exhibit muted morphological innovation and share a rather generalized "riverine cichlid" gestalt, Teleogramma species have highly specialized anatomies. When first described by Boulenger (1899), the genus was erroneously placed in the family Labridae. Myers (1939) and subsequent studies placed it within the Cichlidae, and all noted the highly distinctive nature of these unusual riverine cichlids. Takahashi and Nakaya (2001)  Due in part to their highly autapomorphic morphology, determination of the interrelation¬ ships of Teleogramma within the Cichlidae has proven elusive in morphological analyses (e.g., Stiassny, 1997;Takahashi and Nakaya, 2001). However, a recent multilocus phylogeny (Schwarzer et al., 2014) provides conclusive support for the placement of Teleogramma as nested within the chromidotilapiine lineage of African cichlids. Chromidotilapiines are a primarily riverine clade that originated in West and Central Africa in the late Eocene/early Oligocene (Schwarzer et al., 2014), and therefore represent an early African cichlid radiation with origins long predat¬ ing those of the hyperdiverse, predominately lacustrine, assemblages of eastern Africa.
Prior to the present study, Teleogramma was considered to contain four species (Roberts and Stewart, 1976 During a period of exceptionally low water throughout western Congo in the summer of 2011, a series of highly distinctive specimens were collected in newly exposed rocky outcrops along the lower reaches of the middle Congo River just upstream of Pool Malebo (fig. 2). These specimens were unassignable to any previously described species. Preliminary morphological examination suggested that, although lacking a number of distinctive features of the genus described by Takahashi and Nakaya (2001), these specimens likely represented an undescribed species of Teleogramma, a finding fully corroborated in the present study with both morpho¬ logical and molecular evidence.
In addition to detailed morphological examination, and in order to phylogenetically place the new species within the genus, representatives of all putative Teleogramma species and a chro¬ midotilapiine outgroup were sequenced for two nuclear (SH3PX3 and Ptr) and two mitochon¬ drial (COl and ND2) loci. Model-based phylogenetic reconstructions were undertaken on these molecular data and the results of those analyses provide a comparative framework for the formal taxonomic description of the distinctive new species, Teleogramma obamaorum, provided herein.  (Boulenger, 1899) general habitus of currently recognized species, in lateral aspect, S above, $ below. A, T. brichardi (AMNH 246986); B, T. gracile (AMNH 247261); C, T. depressa (AMNH 239325); D, T. monogramma (AMNH 247806 d, 253127 ?). Scale bars = 1 cm.

Materials and Methods
Morphology: Fourteen standard morphometric measurements and seven meristic counts were taken following Barel et al. (1977). Specimens were carefully pinned flat and photographed on the left side using a Nikon Digital SLR camera with a 60 mm f/2.8 AF Micro-Nikkor lens. Linear measurements were then taken using the open access software ImageJ vl.48 (Schneider et al., 2012), and vertebral and fin-ray counts were taken from radio¬ graphed and/or cleared and stained specimens. Gill raker counts are totals for the first arch and include the raker at the junction of ceratobranchial and epibranchial elements. Lateral¬ line counts exclude the small pored scales on the caudal fin distal to the point of caudal flexion. For comparative purposes, corresponding counts and measurements for the closely related T. monogramma and T. brichardi are provided. Additional specimens of all putative species were examined and cleared and stained using a modified protocol of Taylor and Van Dyke (1985).
Forward and reverse chromatograms were edited and aligned using Geneious R7.0 (Bio¬ matters Ltd., Aukland, NZ). Alignment across all specimens was performed using MUSCLE v3.5 (Edgar, 2004), and best-fit models of nucleotide evolution were determined using JModelt-

Phylogenetic Analyses
Likelihood and Bayesian model-based phylogenetic analyses resulted in the same topology ( fig. 3), and provide strong support for the generic placement and taxonomic validity of Teleogramma obamaorum. The results of this small-scale analysis also provide a preliminary estimate of species relationships with the genus, which is partitioned into two main clades. Within "clade A" the new species, T. obamaorum, is resolved as sister to the Kasai/Lulua-dwelling T.
monogramma, and together these are sister to T. brichardi. We note that T. brichardi, as recog¬ nized herein, is restricted to specimens from the immediate vicinity of the type locality at the fishing village of Kinsuka, located on the left bank of the LCR at the first rapids downstream of Pool Malebo in the Democratic Republic of Congo ( fig. 2).  While support is strong for the monophyly of "clade B" Teleogramma, which comprises the remainder of the LCR populations, branch lengths in this region of the tree are extremely short and the resultant topology highlights conflict between morphology-based taxonomy versus molecular signal. Such short branch lengths, in conjunction with molecular and morphological discord, suggest that rapid and recent divergence, and perhaps ongoing gene flow between incipient and/or young species, may be confounding the molecular signal provided by the four molecular markers used in this study. To further investigate the popu¬ lation dynamics, relationships, taxonomic status, and age of the LCR Teleogramma we are currently analyzing a genomewide sampling of Teleogramma populations utilizing restric¬ tion site associated (ddRAD) markers (Alter et al., in prep.). Despite the inability of our four-locus dataset to entirely resolve the species composition and relationships among the LCR "clade B" Teleogramma, in contrast "clade A" taxa are well supported by these data.
Based on our molecular analyses, and a series of morphological and ecological attributes discussed below, we provide a formal taxonomic description of a new species of Teleo¬ gramma, T. obamaorum, a taxon geographically located between the widely disjunct LCR and Kasai/Lulua populations. T. depressa (Inga) T. depressa (Inga) T. depressa (Inga) T. depressa (Foulakari) T. depressa (Mbudi) T. depressa (Foulakari) T. brichardi (Kinsuka) T. brichardi (Kinsuka) .1 brichardi ( 5A). Four or 5 inner rows of small, slightly recurved, unicuspid teeth clustered on anterior third of dentary; no inner rows distally on lower jaw. Two or three inner tooth rows anteriorly on premaxilla, tapering to single row terminating at two-thirds of length of dentigerous arm. Lower pharyngeal jaw is markedly more robust than in congeners with slightly sinuous, rather than straight, ventral suture (com¬ pare e.g., figs. 6A and B, C). Usually 15-16 moderately robust, unicuspid teeth in posterior row.
Gut Morphology and Diet: Digestive tract short, total length (unraveled but not stretched) ca. 60%-65% SL. Esophagus leads to small bulbous stomach, from which intestine exits left side at transition zone between esophagus and stomach. Proximal descending limb of intestine wide anterior to rostrocaudad loop. Distal limb descending to anus also extremely wide ( fig. 7A). All congeners (e.g., figs. 7B,C) with proximal descending limb of intestine wide-a feature interpreted here as a putative synapomorphy of the genus-whereas distal limb is narrow and similar to condition in other cichlids (Zihler, 1982;Tougas and Stiassny, 2014). Stomach and full length of gut of all specimens contained small, intact shells of two species of rissooidean gastropods (super¬ family Rissooidea) ( fig. 8). In most cases no other food items were present, although few indi¬ viduals also contained some disarticulated remains of ephemeropteran nymphs. ventrally. Although collected while reproductively active, female T. obamaorum also appear to lack the pronounced red flush of color on the belly that characterizes females of other Teleogramma species (Roberts and Stewart, 1976) and most chromidotilapiines (Lamboj, 2004). In preservation ( fig. 4), the base body coloration is dark brownish black, slightly darker dorsally becoming paler ventrally. Unpaired fins are uniformly blackish brown.
Fecundity appears to be low. The largest female of Teleogramma obamaorum examined (63.8 mm SL) contained only 19 ovoid eggs measuring approximately 2 mm in height and 1.8 mm at widest girth and the smallest female (51.9 mm SL) contained 15 eggs of a similar size. In contrast, egg numbers in ripe individuals of similar sized T. brichardi and T. monogramma were higher, averaging 25 to 35. Although some T. obamaorum specimens were "spent," most females contained ripening or fully mature eggs in paired ovaries and in most males' testis development was advanced, indicating that our collections were made during a reproductive period for the species. 2A). At this time a series of rocky outcrops, that in previous (and subsequent) years had been completely submerged, were partially exposed and accessible for sampling and it was only on these that specimens of T. obamaorum were collected (e.g., fig. 2B). On the right bank of the Congo River in the Republic of Congo, and further upstream on the left bank in the Demo¬ cratic Republic of Congo, very few rocky outcrops were located, and most of the shoreline was comprised of sandy or reed-fringed banks, suggesting that the short stretch where all T. obam¬ aorum specimens were collected may represent the entire distributional range of the species. As previously noted, all specimens of T. obamaorum were found at a time of record low water when they were collected upstream of Pool Malebo among rocks newly exposed at the water surface and extending from the shoreline into the main channel of the middle Congo River.
Numerous collections had been made along these shorelines in previous (and subsequent) years when water levels were at standard depths, but no specimens of T. obamaorum were collected. It would appear then that T. obamaorum is likely restricted to deepwater, rocky habitats that typi¬ cally remain submerged well below the water surface, but became exposed and accessible for sampling during the exceptionally low water stand of 2011. It is noteworthy that in these same, rarely exposed habitats, we also collected representatives of three species that had previously been considered to be endemic to rocky outcrops of the LCR (Lowenstein et al., 2011): Chrysichthys helicophagus, Platyallabes tihoni, and Mastacembelus simbi.
Our inference that T. obamaorum is restricted to deepwater rocky habitats is supported by its markedly inflated canals and pores in the laterosensory system of the head, jaws, suspensorium and infraorbital series. These are features associated with enhanced sensory acuity in diverse lineages of deepwater lake cichlids (Fryer and lies, 1972;Webb et al., 2008), and not found in other species of Teleogramma.
Examination of gut contents suggests that Teleogramma congeners are almost exclusively insectivorous, feeding on aquatic larvae and nymphs, primarily ephemeropterans but also, to a lesser extent, trichopterans, plecopterans, and chironomids extracted from rock surfaces and interstices. In contrast, the diet of T. obamaorum consists almost exclusively of rissooidean gastropods ( fig. 8). It seems probable that at depth, Teleogramma obamaorum utilizes its enhanced laterosensory system for prey location (Webb et al., 2014), selectively extracting the small gastropods from among rocks deep in the water column. While all shells retrieved from guts appear intact, it is likely that T. obamaorum utilizes its robust pharyngeal jaw apparatus and relatively massive LPJ dentition to manipulate and slightly fracture the shells before passing them to the digestive tract for enzymatic breakdown of soft tissue. Because the shells are not dismantled during processing and transport, and must be accommodated along the entire length of the gut, the characteristically expansive distal limb of the intestine observed in T. obamaorum ( fig. 7A), but absent in insectivorous congeners, may represent an additional tro¬ phic adaptation serving to accommodate empty snail shells prior to extrusion.
As noted above the lack of sexual dichromatism in T. obamaorum is distinctive within the genus. The lower and middle Congo River carry a heavy sediment load, with high turbidity and intense humic coloring derived from the upstream Cuvette Centrale (Roberts and Stewart, 1976), and light attenuates rapidly from between 0.5 to 1 m below the surface (personal obs.).
Under these circumstances, color vision at depth is likely to be extremely limited, and we speculate that this difference in habitat depth occupancy may account for the lack in T. obam¬ aorum of the sexual dichromatism common to all its congeners (Roberts and Stewart, 1976).
The discovery of a new, ecomorphologically specialized Teleogramma species from the lower reaches of the middle Congo River underscores the highly specialized nature of this unusual clade of riverine cichlids. While the range of phenotypic and ecological diversity exhib¬ ited by members of the hyperdiverse cichlid assemblages of the East African Great Lakes con- NO. 3831 tinues to inspire interest in the processes and mechanisms underpinning these remarkable lacustrine radiations (e.g., Santos and Salzberger, 2012;Brawand et al., 2014), considerably less attention has been focused on the evolutionary dynamics of riverine cichlids (but see Markert et al., 2010;Schwarzer et al., 2011). However, the high degree of morphological specialization described in the present study, in addition to previous work highlighting the importance of standing variation in ancestral riverine lineages (Brawand et al., 2014), suggest that a deeper understanding of riverine cichlids, particularly those exhibiting specialized ecologies and mor¬ phologies, will yield unique insights into diversification processes in the Cichlidae. In particu¬ lar, an in-depth analysis of evolutionary dynamics in riverine cichlids such as Teleogramma may help determine whether genomic (e.g., gene duplication) and ecomorphological features associated with lacustrine radiations are common to cichlids across environmentally disparate habitats and of varying evolutionary ages. Ongoing efforts to develop genomic resources for several African riverine genera (including Teleogramma and Lamprologus) hold great potential to improve understanding of the genomic mechanisms that generate the extraordinary mor¬ phological diversity found among cichlids.