First record of Foulassi Screeching Frog , Arthroleptis adelphus ( Perret , 1966 ) ( Anura , Arthroleptidae , Arthroleptinae ) , from Nigeria , with notes on its phylogenetic position

We report the first known occurrence of the Foulassi Screeching Frog, Arthroleptis adelphus (Perret, 1966), from Nigeria. A specimen of A. adelphus was collected during herpetological survey work conducted in Cross River National Park, south-eastern Nigeria. Morphometrics and mitochondrial 16S rRNA gene confirm identity of the specimen. Matrilineal genealogy reveals a sister relationship of A. adelphus from Nigeria with individuals from south-western Cameroon. Genetic analysis further shows geographic structuring and divergence among populations of A. adelphus from the Guineo–Congolian forest region. We offer updates to the IUCN geographic range of A. adelphus.


Introduction
Species of the genus Arthroleptis Smith, 1849 are found in tropical sub-Saharan Africa (Blackburn 2008, Frost 2019).Commonly referred to as screeching frogs or squeakers, they are mostly terrestrial breeders, feeding on a range of arthropods including ants and termites (Blackburn 2008).Currently, 48 species are known to occur in sub-Saharan Africa (Frost 2019).In Nigeria, this genus is represented by 3 species: Arthroleptis palava (Blackburn, Gvoždík & Leaché, 2010), Arthroleptis poecilonotus (Peters, 1863), and Arthroleptis variabilis (Matschie, 1893) (AmphibiaWeb 2019).There has been no record of Arthroleptis adelphus (Perret, 1966) in Nigeria, although it occurs nearby in the forests of southern Cameroon, Equatorial Guinea (including Bioko), Gabon, and possibly southwestern Central African Republic and northwestern Republic of the Congo (Frost 2019).Arthroleptis adelphus is known to inhabit leaf litter within lowland forests, often avoiding marshy areas (Burger et al. 2006, IUCN 2014, Larson and Zimkus 2018).Although this species is classified by the IUCN as Least Concern, forest loss associated with agriculture, logging and human settlement could negatively impact its survival and distribution (IUCN 2014).
Herein, we report the first record of A. adelphus in Nigeria and offer updates to the IUCN geographic range for A. adelphus.

Methods
Specimen collection.Ethical approval for the field survey (NPH/GEN/121/XXV/461) was obtained from National Park Service, Abuja, Nigeria.The herpetological survey was conducted in Cross River National Park (CRNP) (Fig. 1) from 18 June to 15 December 2018.Using visual encounter survey and opportunistic observation, 5 individuals of A. adelphus were observed in the field by BEA and JYU during the day.However, only 1 juvenile individual was collected as the others escaped before capture.Information such as habitat type and geographic coordinates were documented.The specimen was euthanized humanely with hydrous chlorobutanol.A tissue (muscles) sample was collected and stored in 95% ethanol.Subsequently, the voucher specimen was fixed with 4% formalin and preserved in 75% ethanol for long-term storage.The voucher and tissue samples were deposited in the museum of the Department of Zoology, University of Ibadan, Ibadan, Nigeria.

Molecular laboratory protocol.
Total genomic DNA of the ethanol preserved tissue was extracted following phenol-chloroform extraction procedure (Sambrook and Russell 2001).Matrilineal genealogy was inferred using mitochondrial gene fragment 16S rRNA, which is commonly used as a DNA barcoding marker for African anurans (Nneji et al. 2019).An approximately 540 bp segment of 16S rRNA gene was amplified with primers designed by Bossuyt and Milinkovitch (2000).The PCR cycle profiles were as follow: 5 min initial denaturation at 94 °C, followed by 35 cycles of 1 min at 94 °C, annealing for 45 s at 55 °C, extension for 1 min at 72 °C; final extension for 10 min at 72 °C.Purified PCR products were directly sequenced in both forward and reverse directions with an automated DNA sequencer (ABI 3730).The nucleotide sequence was viewed and confirmed manually using SeqManTMII (DNASTAR Lasergene 7).Furthermore, the amplified sequence was submitted to BLAST search in NCBI (https://blast.ncbi.nlm.nih.gov/Blast.cgi) to verify for sequence similarity.

Genetic data analysis.
To further confirm the identity of the specimen and examine its phylogenetic position, our sequence was compared with others obtained from GenBank.A total of 101 16S rRNA sequences of 7 species belonging to the genus Arthroleptis from West and Central Africa were downloaded as follows: A.  A1).Sequences of 3 closely related species were downloaded to include as outgroup taxa: Arthroleptis formosus (Rödel, Kouame, Doumbia Our herpetological survey of CRNP discovered that Arthroleptis adelphus occurs within the lowland forest of Erokut Camp of Oban East Range, CRNP (Fig. 2A).Individuals of Arthroleptis adelphus were commonly encountered within the leaf litter of lowland forest and on vegetation 0.5-1.0m above the ground.The specimen of Arthroleptis adelphus from CRNP represents the first record from Nigeria, extending this species' geographic distribution west by approximately 149.9 km from its nearest location in south-western Cameroon.The voucher specimen was deposited in the museum of the Department of Zoology, University of Ibadan, Nigeria, under reference number CRNPA0049.The 16S rRNA sequence was submitted to GenBank under accession number MK377154.
Identification.The voucher specimen was morphologically identified as a member of the genus Arthroleptis by the presence of moderate pedal webbing, and the absence of tarsal tubercle, outer metatarsal tubercle, heel tubercle (located at the proximal end of the tarsus) and circummarginal groove at manual and/ or pedal digit tips.The individual was identified as juvenile because of its small body size and lack of secondary sexual characteristics.The BLAST search for 16S rRNA sequence of our specimen showed 99% DNA sequence similarity with A. adelphus FJ151093 and FJ151092 from Cameroon.Sequence data showed that A. adelphus CRNPA0049 from Nigeria clustered genetically in a clade consisting of other A. adelphus from Central Africa (Figure 3).ML analysis revealed that CRNPA0049 formed sister group relationship with specimens FJ151093 and FJ151092 from Ejagham Forest Reserve, Southwest Cameroon (ML bootstrap proportion = 97%, Fig. 3).
Distribution, natural history and conservation.Arthroleptis adelphus is now known from lowland forests in southeastern Nigeria.Its documented range now extends from lowland forests of south-eastern Nigeria to southern Cameroon, Equatorial Guinea (including Bioko), and Gabon.The most common syntopic anurans in CRNP were Sclerophrys gracilipes (Boulenger, 1899) and Sclerophrys maculata (Hallowell, 1854).Considering the relatively large geographic distribution of A. adelphus across Central Africa, we do not consider A.  adelphus to be threatened.However, increased deforestation and agricultural practices in the region pose threats to this species.Additional field studies appear necessary to document population density and status.

Discussion
Our study documents the occurrence of A. adelphus from the lowland forest of south-eastern Nigeria.Previously, its distribution was thought to span across southern Cam-  Africa.This calls for a concerted approach for the conservation of this species.
The phylogeny generated as part of this study was not only informative with respect to confirmation of the identity of our specimen from CRNP, but also had implications for the possibility of cryptic diversity within A. adelphus, as proposed by Deichmann et al. (2017).Divergence among populations of A. adelphus is evident, and CRNPA0049 is more closely related to specimens from south-western Cameroon than it is to specimens from elsewhere (Fig. 3).The type locality for A. adelphus is "Foulassi", near Sangmelima, South Cameroon (Frost 2019), and CRNPA0049 was collected near the type locality.This, therefore, provides additional confirmation regarding the validity of our specimen as A. adelphus.Thus, we offer recommendations for the IUCN update of the geographic range of this species.

Figure 1 .
Figure 1.Map showing the sampled locality, Cross River National Park, Cross River, Nigeria.The new record of Arthroleptis adelphus was observed and collected from locality indicated by a red star.

Figure 2 .
Figure 2. A. Habitat where Arthroleptis adelphus CRNPA0049 was collected.B. CRNPA0049 in life.C. Ventral view of the preserved specimen.

and
Rödel 2005, Ernst et al. 2006), and the destruction of habitat poses devastating effects on the population status and survival of A. adelphus across Central and West

Figure 3 .
Figure 3. Matrilineal genealogy.A. Collapsed ML phylogram of some Central and West African species of the genus Arthroleptis inferred based on mtDNA 16S rRNA.B. Expanded ML Phylogram showing the matrilineal relationships within the Arthroleptis adelphus species group.Numbers at each node represent the ML bootstrap values; nodes with bootstrap values above 50% are shown.The new record of A. adelphus from Nigeria is in red.