In June 2007, a previously undescribed monkey known locally as “lesula” was found in the forests of the middle Lomami Basin in central Democratic Republic of Congo (DRC). We describe this new species as Cercopithecus lomamiensis sp. nov., and provide data on its distribution, morphology, genetics, ecology and behavior. C. lomamiensis is restricted to the lowland rain forests of central DRC between the middle Lomami and the upper Tshuapa Rivers. Morphological and molecular data confirm that C. lomamiensis is distinct from its nearest congener, C. hamlyni, from which it is separated geographically by both the Congo (Lualaba) and the Lomami Rivers. C. lomamiensis, like C. hamlyni, is semi-terrestrial with a diet containing terrestrial herbaceous vegetation. The discovery of C. lomamiensis highlights the biogeographic significance and importance for conservation of central Congo’s interfluvial TL2 region, defined from the upper Tshuapa River through the Lomami Basin to the Congo (Lualaba) River. The TL2 region has been found to contain a high diversity of anthropoid primates including three forms, in addition to C. lomamiensis, that are endemic to the area. We recommend the common name, lesula, for this new species, as it is the vernacular name used over most of its known range.

Funding: The research was supported by Arcus Foundation ( http://www.arcusfoundation.org/ ), United States Fish and Wildlife Service ( http://www.fws.gov/grants/ ), a grant from Edith McBean, Abraham Foundation ( http://abrahamfoundation.org/cms/ ), Margot Marsh Biodiversity Foundation Grant, and a Gaylord Donnelley Environmental Postdoctoral Fellowship from the Yale Institute for Biospheric Studies ( http://www.yale.edu/yibs/programs_donnelley.html ). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Copyright: © Hart et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Citation: Hart JA, Detwiler KM, Gilbert CC, Burrell AS, Fuller JL, Emetshu M, et al. (2012) Lesula: A New Species of Cercopithecus Monkey Endemic to the Democratic Republic of Congo and Implications for Conservation of Congo’s Central Basin. PLoS ONE 7(9): e44271. https://doi.org/10.1371/journal.pone.0044271

In this paper, we describe and name the new guenon species and discuss its relationship with its nearest congener and sister species, Cercopithecus hamlyni. This discovery adds a new species to the previously monotypic and poorly known hamlyni species group, and expands our understanding of this unique, semi-terrestrial lineage within the guenon radiation. It furthermore highlights the biogeographic significance and importance for conservation of the eastern interfluvial region of the Congo River’s central basin, known as the TL2 landscape, from the upper Tshuapa through the Lomami River Basin to the Congo (Lualaba) River ( Fig. 1 ). This previously little surveyed forest region is shown to have high taxonomic richness and endemism of anthropoid primates and represents an important area for conservation of Central African forest faunas.

Distribution of Cercopithecus lomamiensis (sp. nov.) and its sister species, C. hamlyni (left), and locations of specimens and observations of C. lomamiensis (right). Outside of DRC, C. hamlyni occurs only at Nyungwe Forest National Park, Rwanda. The TL2 region extends from the upper Tshuapa River, across the Lomami River to the Lualaba River. See Table SI for details of specimens cited.

Discoveries of new African primate species are rare but significant events that clarify taxonomic and evolutionary relationships and highlight important regions of biodiversity for conservation. Here we report the scientific discovery of a new primate species, Cercopithecus lomamiensis, sp. nov., found during field surveys in a remote area of the middle Lomami Basin in central Democratic Republic of Congo (DRC) ( Fig. 1 ). C. lomamiensis represents only the second new species of African monkey to be discovered in the past 28 years. The new species is a member of the tribe Cercopithecini, commonly referred to as guenons, which represents the most speciose clade of extant African primates. Guenons are endemic to sub-Saharan Africa and occupy a range of habitats from wooded savannas to closed forest [1] . The highest diversity of guenons occurs in closed forests in Central and West Africa where species utilize different canopy levels, including the forest floor [2] , and exhibit considerable dietary flexibility, exploiting a diversity of leaf-, insect- and fruit-eating rainforest niches [3] . Geographical and behavioral barriers have been potentially important in guenon speciation [4] , [5] , and the distribution and relationship of species in related clades provides insight into the biogeographic history of Central African faunas and the evolution of key behavioral and ecological traits.

In addition, this published work and the nomenclatural acts it contains have been registered in ZooBank, the proposed online registration system for the ICZN. The ZooBank LSIDs (Life Science Identifiers) can be resolved and the associated information viewed through any standard web browser by appending the LSID to the prefix “ http://zoobank.org/ ”. The LSID for this publication is: urn:lsid:zoobank.org:pub:7D4F3CF8-1C5C-4DC2-9F49-6DF2A1CDE217. This publication has been deposited in the following digital archives: PubMed Central, LOCKSS, Florida Atlantic University Institutional Repository.

The electronic version of this document does not represent a published work according to the International Code of Zoological Nomenclature (ICZN), and hence the nomenclatural acts contained in the electronic version are not available under that Code from the electronic edition. Therefore, a separate edition of this document was produced by a method that assures numerous identical and durable copies, and those copies were simultaneously obtainable (from the publication date noted on the first page of this article) for the purpose of providing a public and permanent scientific record, in accordance with Article 8.1 of the Code. The separate print-only edition is available on request from PLOS by sending a request to PLOS ONE, 1160 Battery Street, Suite 100, San Francisco, CA 94111, USA along with a check for $10 (to cover printing and postage) payable to “Public Library of Science”.

For each encounter with C. lomamiensis, observers noted the canopy position (vegetation stratum) and behavior of each visible animal when it was first detected. Stratum height position was recorded as: ground = 0 m; understory = >0 m and <3 m; mid-story = 3–20 m; canopy = >20 m. Behavior was recorded as stationary, including grooming and resting; feeding, including both ingesting and searching for food; slow movement, defined as movement where foraging was not detected; and rapid movement or flight. Animals still visible after 3 minutes were scanned a second time for position and behavior.

During survey periods, data were collected daily, from 6∶30 to 14∶00 by two experienced observers moving along the area’s permanent transect grid. Observers recorded the time and GPS waypoint for all primate groups encountered (seen and heard, or heard but not seen). For groups that were seen, we estimated the perpendicular distance from the estimated center of each to the transect line, counted the number of animals seen by species, and estimated the number of additional animals that could not be counted accurately. For groups that were heard but not seen, we recorded the occurrence of species based on vocalizations.

We collected data on C. lomamiensis behavior on a pre-established path grid that totaled 64 km in the Losekola Study Area. We collected data over four sampling periods lasting from 10 days to three weeks from March 2008 to April 2010. Surveys were conducted in an area of diverse mature upland forest types bisected by small streams with no recent history of human settlement and little previous incidence of hunting. The Losekola Study Area was protected from all hunting since January 2008.

Trained teams familiar with the calls of all the primates in the TL2 region conducted vocalization surveys between 13 March and 4 December 2009. Vocalization surveys were conducted at a total of 117 survey posts distributed systematically in six, 30 km×30 km blocks that had been previously selected for intensive large mammal inventories (18–22 vocalization surveys per block). The survey area included locations within C. lomamiensis’ known range and adjacent forest east of the Lomami River and west of the Tshuapa River ( Fig. 1 ). Listening posts were always ≥200 m from survey bivouac camps. Observers arrived at listening posts before first light and remained quiet and concealed during the survey. Observers recorded the species, time, compass direction, and relative distance of the calls for all primates heard between 06∶00–06∶30 in one of three distance categories: proximate: callers or their movements seen from the listening post; far: callers not visible but calls had high clarity and amplitude; and remote: all other discernible calls. For each sampling session, we estimated the minimum number of individual callers of each species using the direction and distance of recorded calls.

We made recordings with a Marantz PMD 660 digital recorder equipped with a Sennheiser condenser microphone module (K6/K6P). All recordings were digitized with Raven 1.3 (Cornell Laboratory of Ornithology, Ithaca, New York) using a 1024-point fast-Fourier transform, Hanning window function. We used the resulting spectrographic displays to extract direct measurements of 11 spectral and temporal parameters and five calculated derived measures ( Table S6 ).

We recorded vocalizations of C. lomamiensis on the Losekola Study Area (S 1.38461°, E 25.03749°) between 1 April–25 April 2009 and of C. hamlyni near Epulu (N 1.40200°, E 28.57709°) in the Central Ituri Forest between 20 February and 3 March 2009. We concentrated recording sessions between about 05∶45 and 06∶30, a time when both species exhibit a marked increase in vocal behavior. Observers arrived at recording sites before dawn and remained stationary (and presumably undetected by the animals) throughout recording sessions.

The results of the four separate BEAST runs of both C. lomamiensis and C. hamlyni datasets were checked for adequate mixing and convergence using Tracer 1.5. Following successful convergence, BEAST tree files were combined using LogCombiner with burnins of 10,000 (TSPY) and 25,000 (Xq13.3 homolog). The combined sample of trees was summarized using TreeAnnotator 1.5.3 [26] before being visualized with Fig Tree 1.3.1 [26] .

We used the BEAST 1.5.3 software package [26] to obtain estimates of dates of molecular divergences. This Bayesian program allows the inference of divergences despite significant rate variation among lineages. The XML input files were generated using BEAUti 1.5.3. The substitution models used in the BEAST analysis were again those inferred by MODELTEST (HKY for TSPY, GTR for Xq13.3 homolog), with base frequencies and alpha values estimated from datasets. The uncorrelated lognormal relaxed clock model was employed, with the rate of molecular evolution estimated from the data [27] . The analysis was started using the tree inferred by MRBAYES and with the tree prior set to the birth-death model. Operators were auto-optimized, and the analysis was run on Cornell University’s CBSU cluster ( http://cbsuapps.tc.cornell.edu/index.aspx ) with four repetitions of 20,000,000 (TSPY) or 50,000,000 (Xq13.3 homolog) MCMC generations logged every 1,000 generations. Several nodes were given priors based on estimated divergence (see Text S1 for additional methods on divergence date calibration).

Bayesian analyses of both datasets were conducted using MRBAYES 3.11 [23] , [24] with the molecular evolutionary models inferred by MODELTEST. Each dataset was run twice with four chains for 1,000,000 generations and sampled every 100 generations. Sump and sumt burnins were 2500. Adequate mixing of chains and convergence of runs were verified using Tracer 1.5 [25] .

Maximum likelihood analyses using the appropriate models of molecular evolution were conducted first using PAUP* 4.0b10 [21] and then GARLI 0.951 [22] , with 50 and 500 bootstrap replicates, respectively. Trachypithecus cristatus was used as the outgroup for the TSPY analysis, and T. obscurus for Xq13.3 homolog. GARLI allows much more rapid estimation of likelihood values and was therefore used to generate higher numbers of bootstrap replicates. Recommended default settings were used for the genetic algorithm implemented in GARLI.

The novel TSPY and Xq13.3 homolog sequences of C. lomamiensis and C. hamlyni were each added to datasets consisting of sequences of other cercopithecids taken from GenBank ( Table S2 ). Both datasets were aligned with ClustalW2 [19] and checked by eye. Novel sequences developed in this study were archived in GenBank ( Table S2 ). Alignments for each gene are provided in Matrix S1 and S2 as fully executable Nexus files. Appropriate models of molecular evolution were inferred using hierarchical likelihood ratio tests implemented in MODELTEST 3.6 [20] . The HKY + G model with an alpha value of 0.7748 was inferred for TSPY; TrN + G with an alpha value of 0.9416 was estimated for Xq13.3 homolog.

We used a phylogenetic analysis in a comparative context to address the question of species vs subspecies status for the C. lomamiensis individuals. If the divergence date calculated between the C. lomamiensis lineage and its sister lineage is equal to, or greater than, estimates calculated between recognized species within other cercopithecin species groups, it provides a strong argument for equivalent species-level status of the C. lomamiensis lineage. The TSPY and X-datasets were not combined for these analyses. X- and Y-loci differ in significant parameters, including effective population size and mode of inheritance, and are therefore known to follow unique evolutionary trajectories [17] . Moreover, earlier studies have shown that while the X-locus surveyed here is evolving in a clock-like fashion in cercopithecins [11] , the TSPY gene is not [18] .

Amplified products were cleaned with exonuclease I and shrimp alkaline phosphatase [16] and cycle-sequenced using BigDye chemistry (Applied Biosystems, Foster City, CA). Cycle-sequence products were cleaned via ethanol precipitation and analyzed using an ABI 3730 automated DNA sequencer. Complementary strands were sequenced as a proofreading check of the data. The sequence reads from each amplicon were processed and assembled into a single contig using the program Sequencher v4.8 (Gene Codes Corp.).

We amplified and sequenced a ∼2.2 kb segment of the Testis-Specific Protein, Y-chromosome (TSPY) using primers and protocols described by Tosi et al. [14] . TSPY is a multigene family located in the non-recombining portion of the Y-chromosome and is believed to have a function in spermatogonial proliferation [15] . Data already collected on this gene family suggest that it is maintained by a mechanism of concerted evolution in cercopithecine monkeys [11] , [14] .

We assembled a ∼4.6 kb contig of X-chromosomal DNA from three overlapping amplicons for the newly surveyed C. hamlyni and C. lomamiensis individuals, using primers and protocols described by Tosi et al. [11] . The surveyed region is homologous to a portion of human Xq13.3 and consists solely of intergenic DNA [12] . This is a region of low recombination in both humans [13] and cercopithecins [11] and therefore unlikely to contain substantial reshuffling of ancestral DNA sequences, making it an excellent locus for phylogenetic study.

We digitized the landmarks on each cranium using a Microscribe G2X digitizer (Immersion Corp). The individual crania were immobilized in a bed of Play-Doh, and the landmarks were digitized in two sets–one superior view and one inferior view. All bilateral landmarks were digitized on the right side of the cranium. As reference points, five landmarks were digitized in both views. Subsequently, the two sets of landmarks were combined using the program DVLR v. 0.4.9 [8] to obtain a single set of 23 three-dimensional coordinates for each cranium. We imported the landmark data into the software package Morphologika [9] , and performed a Procrustes superimposition analysis on the entire sample of C. lomamiensis (n = 3) and C. hamlyni (n = 7) crania. We then used a principal components analysis (PCA) of the Procrustes superimposition to identify the major axes of cranial shape among all of the crania. Pelage and skin coloration are described using standard color references [10] .

Craniodental linear measurements were taken using digital calipers and recorded to the nearest tenth of a millimeter ( Tables S3 , S4 ). In addition to standard caliper measurements, we used 3-D geometric morphometric techniques to compare cranial shape of C. lomamiensis and C. hamlyni. The 23 landmarks chosen to capture the overall shape of the cranium are listed in Table S5 and include Type 1, Type 2, and Type 3 landmarks [6] ; these landmarks included those used by Fleagle et al. [7] in their larger study of primate cranial diversity, and additional cranial landmarks deemed to be repeatable and informative.

All C. lomamiensis skin and skeletal specimens are housed in the Yale Peabody Museum (YPM), New Haven, CT. Adult C. hamlyni specimens were examined at the YPM and the American Museum of Natural History (AMNH), New York, NY. See Table S1 for details on the specimens examined.

Seven specimens of C. lomamiensis and eight specimens of C. hamlyni were used for analyses and descriptions of C. lomamiensis and C. hamlyni ( Tables S1 , S2 ). Specimens collected in the field included freshly killed animals acquired from local hunters, animals killed by predators (including kills by leopards, Panthera pardus, or crowned eagles, Stephanoaetus coronatus) and one skin snip from a monkey captured locally and kept as a captive in a village near the species’ range. We used GPS to record locations where specimens were recovered in the field; when exact location of specimen origin was not possible (e.g., location based on hunter reporting), locations were estimated to the nearest settlement or geographic feature. We took information on the provenance, history and care of all captive animals seen. We took photographs of all specimens and captives, and recorded standard field measurements (total length, tail length, length of hind foot, length of ear pinnae, and body mass) wherever possible. Duplicates of tissue samples were stored in RNAlater (Applied Biosystems, Foster City, CA) and 95% ethanol and stored in the field in cool, dark locations until transferred to the laboratory.

The Congolese Wildlife Authority (Institut Congolais pour la Conservation de la Nature, ICCN) issued permits to the TL2 (Tshuapa, Lomami, and Lualaba) Project for all sites where biological samples and field observations were made. The ICCN is the governmental authority that has jurisdiction over the wildlife of this territory. Institutional Animal Care and Use Committees (IACUC) approval was not required for the noninvasive behavioral observations and biological samples of wild monkeys used in this study. IACUC protocols were followed for the collection of one skin snip specimen from a captive monkey. For the specimens collected from hunted animals, we obtained approval from the hunters to use these samples and no animal was hunted for the purpose of research. We acquired specimens only opportunistically in villages outside of the forest and we did not request samples from all lesula available to avoid targeting this species. When we encountered captive monkeys in villages, we photographed them with permission from the owner. We advised owners on the monkeys’ care and discouraged owners to acquire wild animals as captives. All the necessary exportation and importation permits were acquired by CITES, Centers for Disease Control and Prevention and the U.S. Fish and Wildlife Services.

Results

1. Discovery The scientific discovery of Cercopithecus lomamiensis was made in June 2007 when field teams saw a captive juvenile female of an unknown species at the residence of the primary school director in the town of Opala (S 0.50721°, E 24.22713°). The school director identified the animal as a “lesula” a vernacular name we had not recorded before, and said that it is well known by local hunters. He reported that he acquired the infant about two months earlier from a family member who had killed its mother in the forest near Yawende, south of Opala and west of the Lomami River (S 0.99772°, E 24.29810°). We took photographs of the animal and made arrangements for its care. We observed and photographed this animal regularly over the next 18 months. Subsequent searches in Opala and in the Yawende area turned up other male and female captive juvenile lesula; all were photographed and some monitored for several months afterwards. Our first observation of the species in the wild was in the Obenge area (S 1.38461°, E 25.03749°) in December 2007 where the species is well known by local hunters.

Differences in Cranial, Skin and Pelage Traits between C. lomamiensis and C. hamlyni Overall, the olive-maned C. hamlyni is darker, more somber and less vividly marked than C. lomamiensis whose grizzled blond mane, prominent amber dorsal patch, and buff upper ventrum contrast with black legs and lower ventrum. The crown hairs of C. lomamiensis are generally not offset by a contrasting diadem as is often seen in C. hamlyni. The pale facial skin and ear pinnae of C. lomamiensis are distinguished from the dark facial skin and pinnae of C. hamlyni. The nose stripe is typically a yellowish/cream color and often indistinct in C. lomamiensis rather than the clear white of C. hamlyni. Typically, there are 3–4 bands on the dorsum hairs of C. lomamiensis and 4–5 bands on the hairs of C. hamlyni [28]. The gray/silver hairs on the thighs of C. lomamiensis do not extend as far distally as in C. hamlyni. The tail of C. lomamiensis is amber at its base, black over most of its length and lacks a terminal tail tuft. The tail of C. hamlyni is gray from the base to over 3/4 of its length and is only black at its tufted terminal tip (Fig. 4). The juvenile pelage of C. lomamiensis is distinctively paler and blonder than that of C. hamlyni. Juvenile C. lomamiensis have pale facial skin in contrast to the darker facial skin of C. hamlyni, and they show the beginnings of the amber dorsal patch even when immature (Fig. 6). The cranium of C. lomamiensis is most obviously distinguished from C. hamlyni by its significantly larger orbits, greater degree of occipital flexion, narrower interorbital breadth, and narrower calvarium (Fig. 5, Table S4). C. lomamiensis has significantly larger incisors, upper and lower M2s, and upper M3s than C. hamlyni (Table S4). In addition, a distinct bump or prominence is typically present at or around nasion in C. hamlyni, but is absent in the type specimen of C. lomamiensis.

4. Vocalizations The dawn boom chorus is the most conspicuous vocalization of both C. lomamiensis and C. hamlyni and the only call regularly heard in free-ranging animals. We consistently heard booms of C. lomamiensis around dawn and very little during the remainder of the day. We heard occasional booms by C. lomamiensis at night, most often shortly after dark. Though we did not identify the sex of callers, they were almost certainly male; several other Cercopithecus species (e.g., C. campbelli, C. neglectus, C. mitis, C. wolfi) make similar boom calls utilizing a laryngeal air sac that is substantially larger in males and in no species do females produce such a call [29]. We collected 49 useable recordings of ‘descending booms’ by C. hamlyni (n = 43) and C. lomamiensis (n = 6). No other call types were recorded. Exemplars of booms by C. hamlyni and C. lomamiensis, filtered for calls of birds and insects and other ambient sounds are provided in Audio S1 and S2. The booms of C. lomamiensis and C. hamlyni are low frequency, tonal calls clearly distinguished from booms in other Cercopithecus species by their longer duration and descending frequency from beginning to end. In both species, booms were given in bouts of 2–3 calls and different animals often called in overlapping sequences, suggesting contagion or potentially antiphonal calling. The booms of both species consist of a single dominant frequency band with no discernible harmonic overtones or formants (Fig. 8). Booms have a mean starting frequency of 215 Hz in C. lomamiensis and 205 Hz in C. hamlyni and a mean end frequency of 199 Hz in C. lomamiensis and 188 Hz in C. hamlyni. Call duration averaged 0.35 sec in C. lomamiensis and 0.37 sec in C. hamlyni. The calls differed significantly (Welch Two Sample t-test, n = 49, p<0.05) on two of 16 measured acoustic parameters: High frequency and 3rd Quartile Frequency Differences. Three other parameters (End Frequency, Start Frequency, and 1st Quartile Frequency) were nearly significant (range: p = 0.054–0.065) (Table S8).

5. Ecology We found C. lomamiensis in mature upland evergreen humid forests, including mixed forests and forests dominated by Gilbertiodendron dewevrei. We recorded C. lomamiensis less often in regenerating forests around settlements. We never recorded C. lomamiensis in seasonally inundated forests or in the gallery forests in the savannas south of the main forest block. C. lomamiensis is shy and was the least frequently seen of all primates recorded on large mammal surveys (19 observations of C. lomamiensis out of a total of 223 visual observations of primate groups). Vocalization surveys indicate that C. lomamiensis is more abundant than visual sightings on transects would suggest. We recorded C. lomamiensis boom calls at 34 of 70 dawn listening posts surveyed within the species’ known range between the Lomami and upper Tshuapa Rivers. We surveyed an additional 47 listening posts outside the known range, east of the Lomami River and west of the Tshuapa River and recorded no C. lomamiensis vocalizations. Multiple individuals were recorded calling at 27 of 34 (79.4%) sites where C. lomamiensis vocalizations were heard. Of the 27 posts with multiple animals calling, two vocalizing individuals were recorded at 17 posts; three vocalizing individuals were recorded at 4 posts; 4 vocalizing individuals at 5 posts; and 5 vocalizing individuals were recorded at one post. When multiple animals vocalized, it was not possible to determine if the animals belonged to different groups or were members of a single dispersed group. Of 78 calling individuals, five callers were classed as proximate. Two proximate vocalizing animals were seen on the ground; three others were detected by moving vegetation, either on the ground or in the understory. All other vocalizing animals were classed as far or remote. We found no evidence for seasonal variation in calling rates; however, we found marked differences in the occurrence of calling in different portions of the species known range. We recorded vocalizations at 11 of 40 (27.5%) listening points in the southern third of the range. In contrast, vocalizing animals were recorded at 16 of 19 listening points in the region of the Losekola Study Area and at 8 of 11 listening points in the northern portion of the species range. These latter two locations are in the Tutu River basin, a tributary of the Lomami River. These apparent differences in occurrence of C. lomamiensis may be due to habitat differences in the areas surveyed. The southern range has lower rainfall with a pronounced dry season and a higher frequency of savanna ecotone, where lesula were never recorded. Closed evergreen forests dominate the northern range of C. lomamiensis, including the Tutu River basin.