Ocrepeira klamt sp. n. (Araneae: Araneidae), a novel spider species from an Andean páramo in Colombia

Herein we describe Ocrepeira klamt sp. n. (Araneae: Araneidae), a new orb-weaving spider species from a Colombian páramo, which was formerly inaccessible for scientific studies due to decades long armed conflicts. Both, phenotypic and molecular data are used to confirm genus affiliation, and the new species is placed into phylogenetic context with other araneid spiders. Morphological characteristics and ecological notes of Ocrepeira klamt sp. n. are reported together with the sequence of the barcoding region of cytochrome c oxidase subunit I (COI) to provide a comprehensive description of the spider, facilitating future identification beyond taxonomic experts. With this study we contribute to the taxonomic knowledge that is required to inventory the hyper diverse yet threatened ecosystem of the Colombian páramos.


Introduction
Biodiversity hotspots, priorities for conservation efforts due to their high number of endemic species, are thought to harbour most of undescribed organisms [1,2]. Colombia is distinguished by accommodating two hotspots, Tumbes-Chocó-Magdalena and Tropical Andes, the latter being recognized for hosting the highest species richness and most endemics worldwide [1]. Yet, many areas remain unexplored due to a decades-long armed conflict, which has diminished only recently. Concerns are growing that these formally inaccessible territories are now more vulnerable to rapid human-induced change [3,4], turning taxonomic inventories, monitoring and conservation initiatives into an urgent matter.
Located in the Tropical Andes are the Colombian páramos, neotropical alpine grassland ecosystems situated between the timberline and permanent snow fields. Characterized by swamps and wet grassland, conspicuous frailejones (Espeletia) and small shrub and forest patches, they are often referred to as 'grassland isles within a sea of cloud forests' [5][6][7]. This

PLOS ONE
PLOS ONE | https://doi.org/10.1371/journal.pone.0237499 August 24, 2020 1 / 17 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 natural isolation and fragmentation has generated their high biodiversity and endemism, which, in combination with the páramos' key role in Colombia's hydrological system and growing disturbance through human activities, gives them particular significance and warrants special conservation efforts [6,7]. Araneae (spiders) are a group of extremely diverse and abundant key predators, present in every terrestrial ecosystem [8][9][10]. They have been suggested as good ecological indicators, i.e. suitable to monitor functional changes in an ecosystem, as they respond readily to alterations in their biotic and abiotic environment [11]. Their utility depends on a profound knowledge on the identity of present species.
The family Araneidae Clerck, 1757 [12] is one of the most diverse spider families, currently including more than 3000 species in 178 genera [13]. Although Neotropical araneids are considered to be well-studied from a taxonomic perspective, as provided by H.W. Levi's monographic revisions (see the complete list of Levi's publications in [14]), several recent publications have revealed undescribed species across different genera [15][16][17][18][19][20]. In the quest to understand and protect biodiversity, uncovering species' identities is a requisite, but it is a task that is rarely encouraged in our current times [21,22]. Additionally, molecular and phenotypic data are seldom reported together, neglecting the potential of such a combined approach [23].
Here, we describe a new spider of the genus Ocrepeira Marx, 1883 [24] (Araneidae: Araneinae), and provide notes concerning its ecology from the Páramo Las Hermosas (Colombia), an area formerly inaccessible due to armed conflicts. To date, this orb-weaving genus includes 67 recognized species [13], which are found exclusively in the Americas and prevailingly inhabit high mountain environments, where the geographic isolation causes a high degree of endemism [25]. In providing the so far third ever sequence of the barcoding gene cytochrome c oxidase subunit I (COI) from the genus Ocrepeira [26,27], and exploring the phylogenetic position of the new species, we complement the morphological description with molecular data.

Study area & sampling
Spider sampling was conducted in an alpine grassland ecosystem in Valle del Cauca Colombia, the Páramo Las Hermosas, La Nevera locality (03˚31'54.8'' N, 76˚04'40.1'' W, elevation 3650 meters above sea level) in November of 2018 (Fig 1). Climatically, the area belongs to the montane, per-humid Holdridge life zone [28,29], with a mean annual temperature of 7.4˚C (calculated according to [30]) and mean annual total precipitations of 2400 mm (based on IMERG multi-satellite precipitation estimates [31] from the years 2014 to 2019). The study area is dominated by tussock grasses, dwarf shrubs and Espeletia, with few bush and tree patches (S1 Fig) [6]. We sampled the spiders through visual search and with a sweeping net, stored them individually in 95% alcohol and transferred them to a freezer (-20˚C). The collection of specimens was performed under the permit #0526 granted by the Autoridad Nacional de Licencias Ambientales (ANLA) to the Universidad Icesi.

Morphological analysis
Taxonomic description follows the format used by Cabra-García & Brescovit [34]. Specimens were examined using a Nikon C-PS stereomicroscope and a Nikon Eclipse Ci compound microscope. In order to visualize female internal genitalia, non-chitinous tissue was digested with pancreatin following the protocol described by Á lvarez-Padilla & Hormiga [35]. We took the photos of preserved specimens and genitalia using a Nikon SMZ-1500 stereomicroscope equipped with a Nikon DS-Ri1 U3 camera and a Nikon Eclipse Ci compound microscope equipped with a Canon T5i camera of the in-house Laboratorio de Imágenes at Universidad del Valle (Cali, Colombia). Extended focal range images were composed using NIS-Elements Basic Research Software version 4.20.03. Morphological measurements were performed using the integrated ruler of a Nikon C-PS stereomicroscope with 13.4 x to 100 x magnification. All morphological measurements are in millimetres. Vouchers were deposited in the arachnological collection of the Museo de Entomología de la Universidad del Valle (MUSENUV) and the Instituto de Ciencias Naturales, Universidad Nacional de Colombia (ICN).

Nomenclatural acts
The electronic edition of this article conforms to the requirements of the amended International Code of Zoological Nomenclature, and hence the new species name contained herein is available under that Code from the electronic edition of this article. This published work and the contained nomenclatural acts have been registered in ZooBank, the 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:5385566B-DCD5-4716-9B7F-5351690D3F4C. The electronic edition of this work was published in a journal with the eISSN: 1932-6203, has been archived and is available from the following digital repositories: PubMed Central, LOCKSS.

Phylogenetic placement
Total DNA was extracted from one male and one female specimen of O. klamt sp. n. (MUSE-NUV-Ar 2092, DNA Vouchers A310 and A311). Thereby, for each specimen four legs were frozen with liquid nitrogen and ground up; the remainder of the spider was kept as a voucher. Subsequently, extractions including a negative control were carried out using the DNeasy Blood & Tissue Kit (Qiagen) including a RNAse (Promega) treatment (40 μg/ml, for 15 min at 37˚C). DNA amplification of the COI locus was performed using existing invertebrate primers [36] adapted for the use with Araneomorphae spiders (HCO2198_spider TAWACYTCDGGRTG HCCAAAAAATCA; LCO1490_spider ATTCWACWAAYCAYAAGGATATTGG). Polymerase chain reactions (PCR) were performed in a 50 μl volume with 0.02 U/μl Taq DNA Polymerase, 1x ThermoPol buffer and 0.2 mM each dNTP (NEB), using 0.2 μM of each primer and between 30 and 50 ng of template DNA. A "touchdown" PCR profile was employed: Denaturation at 95˚C for 30 s was followed by the first cycle set (6 repeats) with 20 s denaturation at 95˚C, 60 s annealing at 48˚C (-1˚C per cycle) and 60 s extension at 68˚C. The second cycle set (39 repeats) consisted of 20 s denaturation at 95˚C, 60 s annealing at 43˚C and 60 s extension at 68˚C, followed by a final extension step of 5 min at 68˚C. PCR products were purified using the Wiz-ard1 SV Gel and PCR Clean-Up System (Promega) and Sanger-sequenced (Eurofins Genomics). Contiguous sequences were assembled using the package consed/phred/phrap [37][38][39][40]. Once assembled, contigs were queried against the online NCBI BLAST database to check for possible contamination from external sources.
The best tree was inferred in a maximum likelihood framework as implemented in IQTREE v.2.0 [48]. ModelFinder [49] was used to select the optimal partition scheme and substitution models ( Table 2). Ten independent runs, including the calculation of the ultrafast Bootstrap

Species boundaries
Ocrepeira klamt was foremost delimited from other species of the genus by comparing its morphological characters of the genitalia with the available literature [25,52]. In addition, genetic distances were used to test for species boundaries within our Ocrepeira sample. The uncorrected intra-and inter-specific COI divergence among the available Ocrepeira species was calculated using MEGA X [53]. Species boundaries were tested using the Automatic Barcode Gap Discovery (ABGD) method [54]. ABGD analyses were carried out with the command-line version of the program, employing the simple distance metric (i.e. p-distance). The data were analysed using two different values for the parameters P min (0.0001 and 0.001), P max (0.1 and 0.2), and relative gap width (0.1 and 0.4), with the other parameters maintained at default values.  Levi, 1993 [25] by the presence of a triangular offset in the base of the median apophysis (Fig 2A and 2C). They are distinguished from the latter by the folded embolus lamella (Fig 2A) and the sharpened apical portion of the paramedian apophysis (Fig 2A and 2C). Females can be easily distinguished from all other Ocrepeira species by the three apical lobes of the median plate ( Fig 3B).

Species description
Description. Male (holotype). Fig 4A-4C. The carapace of the holotype is of khaki coloration, with a taupe brown cephalic region and dusky irregular areas in the thoracic region projecting to the lateral surface. Chelicerae black with the apical portion brown. Labium taupe brown with an anterior greenish rim. Endites walnut brown with greenish rims on their anterolateral margins. Sternum with a taupe brown fringe and a light greenish centre. Legs are taupe brown with buff brown and greenish patches, light coloured rings on the basal portion of femora, tibia, metatarsi and tarsi. The abdomen has two anterolateral and an anterior median hump; coloration of dorsum grey with a robin egg blue tint and a V-shaped beaver brown central pattern, delimited by black markings; anterior hump with Robin egg blue patch;
sclerotized and with a membranous distal fold beneath the embolus. Paramedian apophysis spine-like, connected to the conductor. Radix without median outgrowth. Embolus pointed, with a basal sclerotized sharp projection. Embolus lamella folded apically. Subterminal apophysis rounded apically. Terminal apophysis spine-like. Female (paratype). Fig 4D-4F. The female has a chocolate brown carapace, with similarly coloured chelicerae, labium and endites (the latter two having an anterior white rim). Sternum black. Legs chocolate to taupe brown, with lighter patches in the basal portion of femora, tibia, metatarsi and tarsi. The abdomen has two lateral humps and a small anterior median hump,  and is longer than wide; the central part of the dorsum has a V-shaped chocolate brown pattern, the sides are dirty white and the anterior part of the abdomen shows a median white spot tinted grey-greenish; the venter is chocolate brown in the middle, bordered by two thin white lines, sides are taupe brown. Leg formula 1,2,4,3. For body measurements see Tables 3-5. Epigynum wider than long, sclerotized (Fig 3A-3C). Scape set off from base, longer than wide (Fig 3A and 3C). Median plate as wide as the maximum width of the lateral plates. The median plate with an undulated anterior rim with three lobes. Rounded spermathecae (Fig 3D-3F). Fertilization ducts shorter than copulatory ducts (Fig 3E and 3F). Copulatory ducts with a basal enlargement (Fig 3D and 3E). Variation. Male. Coloration of paratypes lighter than in holotype, carapace dominated by shades of yellow. Tint of dorsum of abdomen can be green, and V-shaped pattern might be indistinct (Fig 5). White lines on the abdomen venter can be absent. Total length varies from 4.60 mm to 4.96 mm, carapace length from 2.75 mm to 3.18 mm (n = 3).
Female. Coloration of females varies from light (shades of yellows) to dark (shades of brown) (Fig 6). Seldom carapace with ruby red tint. Dorsum of abdomen very variable, colorations include: grey, walnut or taupe brown, ruby red, pink or green tint, grey-brown patterned. Venter with or without white lines or spots. Total length from 5.47 mm to 7.11 mm, carapace 2.85 mm to 3.55 mm (n = 9). On average females are 23% larger than males.
Distribution. Known only from the type locality. Ecological notes. As typical for Ocrepeira, O. klamt sp. n. builds orb webs. In shape they range from elliptical to round, and maximum heights and widths of around 26 cm x 21 cm, as well as minimum heights and widths of approximately 18 cm x 13 cm were observed in the field (Fig 7). They usually show a disorderly filled hub region. Webs were found in open, as well as slightly protected habitats (i.e. with nearby vegetation blocking e.g. strong winds) at
heights between 0.4 m and 1.6 m above the ground. Spiders were spotted in their webs exclusively at night time, typically sitting in the hub, whereas during the day they could be collected sweeping the vegetation.

Species identity and phylogenetic placement
Genus identification. The genus Ocrepeira was first mentioned by Marx [24], but it was Levi [25] who authored a comprehensive monograph for the genus, providing an updated

PLOS ONE
New Colombian spider species, Ocrepeira klamt sp. n. diagnosis and a detailed morphological description. Levi [63] also proposed a key to the genera of araneids of the Americas, which allows to set apart Ocrepeira from similar genera considering female and male somatic and genital characters. Based thereupon we placed the new species in the genus Ocrepeira by confirming the presence of the following combination of characters in the examined specimens: carapace wide in the eye region, posterior median eyes facing dorsolaterally, clypeus height equal to one or at most two diameters of the anterior median eyes, abdomen with two anterior humps, pedicel attachment at the anterior half of the abdomen, and straight paramedian apophysis, all of them considered by Levi [66] and Pozonia Schenkel, 1953 [67].
Additionally, we consider the phylogenetic placement in the DNA-based tree (Fig 8), where O. klamt sp. n. clusters together with O. ectypa and O. darlingtoni, albeit with low support. We acknowledge that due to the low support, our genus placement may be considered contentious. Nevertheless, an unequivocal placement would only be feasible after testing Ocrepeira monophyly using a comprehensive taxon sampling and molecular and morphological characters, a task beyond the scope of this paper.
Phylogeny. A recently published comprehensive phylogeny of araneid spiders, which was based on nuclear and mitochondrial genes, placed the species Ocrepeira ectypa as sister to

PLOS ONE
New Colombian spider species, Ocrepeira klamt sp. n. however, differ between the two phylogenetic trees. Specifically, nodes that yielded ultrafast bootstrap values below 70% are inconsistent with results produced by [26]. A combined effect of few molecular markers and terminals in the present study may explain these inconsistencies. Species delimitation. At species level, differences in genitalia structures are generally considered key for alpha-taxonomy due to their rapid evolutionary divergence [68]. When compared to the available scientific literature (i.e. [13,25,52]), both male and female O. klamt sp. n., exhibit a unique set of characters of the reproductive organs, setting them unequivocally apart from other Ocrepeira species (Figs 2 and 3). It is noteworthy that female internal genitalia across Ocrepeira species remain highly understudied. Nevertheless, a comparison of the available data (i.e. dissections of O. darlingtoni in [27]) with our cleared view (Fig 3D-3F) suggests that the morphology of the copulatory ducts could be highly informative to distinguish species.
The ABGD analyses among the available Ocrepeira sequences also suggested O. klamt sp. n. as a separate species. It is worth mentioning that while the low number of sequences per species available for the ABGD method may compromise its performance [54,69,70] have found that this method can yield similar results to other species delimitation tests, despite the low number of sequences per species.
Matching of the sexes and utility of COI. The matching of male and female individuals of an undescribed spider species is often problematic due to considerable sexual dimorphism. As one female O. klamt sp. n. individual was found in its web together with a male, a common species identity can however be inferred. Additional evidence comes from female and male COI sequences, which are identical (Table 6). COI, the so-called barcoding gene, is a useful tool to distinguish species, even those that are difficult to identify in most phyla with morphological taxonomic methods [71]. In the species-rich order Araneae, COI might be particularly helpful, making identification accessible to non-specialists and facilitating the identification of the more abundant juveniles, which cannot be distinguished phenotypically [72]. The utility of COI has been tested and confirmed for different spider families [73][74][75], and has been shown to yield an identification accuracy of 90% in Araneidae [75]. The here reported COI sequence from O. klamt sp. n. thus provides a valuable character to be used in conjunction with morphology for species identification. Being the third ever reported sequence from the genus Ocrepeira [26,27], it adds to the growing reference database of COI sequences for spiders [72,76].
klamt sp. n. the highest recorded species of its genus (compare to [25,52,77]). So far, O. klamt sp. n. is solely known from the type locality, and the island characteristic of páramo ecosystems suggests that the species has a small distribution range, similarly to that of other known Ocrepeira species [6,25].

Conclusion
The discovery of Ocrepeira klamt sp. n. contributes to a steady stream of new floral and faunal descriptions from the Colombian páramos [78][79][80] that is increasing since the diminution of the armed conflict. Due to extremely high speciation rates even in comparison with other ultra-diverse ecosystems of the Tropical Andes, the páramos can be considered a 'hotspot within a hotspot' [81], making them a number one priority for monitoring and conservation efforts. With the description of a novel araneid species from the Páramo Las Hermosas, a region with previously unexplored spider fauna due to its inaccessibility caused by armed conflict, we contribute to the taxonomic knowledge required to inventory, monitor and ultimately protect this important ecosystem. Thereby, the combination of molecular with morphological data facilitates the accurate association between male and female individuals, provides two independent sources of support for genus affiliation and expands the utility of the data.