Draft paper August 2018

Hidden diversity of sand catlet fishes in the rivers of Eastern Africa Victor Van der Meersch1, Martin Genner2

1 AgroParisTech, Paris, France 2 School of Biological Sciences, University of Bristol, Bristol, United Kingdom

Abstract Fish biodiversity within the rivers of eastern African rivers is typically not well documented. This lack of knowledge can hinder effective designation of habitats and species requiring conservation interventions. This study investigates mitochondrial oxidase subunit I (COI) sequences to barcode sand catlets (Zaireichthys sp.) samples from Tanzania, Malawi and Zambia. We used two different methods of species delineation, the General Mixed Yule Coalescent (GMYC) approach and the multi-rate Poisson Tree Process (mPTP), to resolve the presence of at least eight potential species, of which three are not previously recognised on DNA barcode databases, and potentially represent new species. Our results add to a growing literature suggestive of cryptic fish diversity in Eastern Africa, and highlighted the need for further research on stream fishes in that region.

Introduction

The failure to recognise reproductively isolated yet morphological similar “cryptic” species can lead to systematic underestimates of biodiversity. The description of potential cryptic species is important to improve the conservation and natural resource management (Bickford et al., 2007). It can also be useful when considering geographical mechanisms behind population isolation, and when evaluating links between habitat connectivity and genetic divergence. Over recent years DNA barcoding has proved to be an effective method for uncovering hidden species diversity in many species groups (Hebert et al., 2004) including freshwater fishes (Pereira et al., 2013).

The family Amphiliidae is lineage of stream fishes that is widely distributed across sub-Saharan Africa, and containing some 66 described species in 12 genera (*). The “sand catlet” Zaireichthys is one of the more species rich of the amphilid genera, and has a wide distribution across eastern and central Africa. There are currently eighteen reported species in this small bodied and stream-living genus (Frick et al., 2018), of which ten have been described since the beginning of the 21 st century (Roberts, 2003; Seegers, 2008; Eccles et al., 2011). Many of the described species have narrow biogeographical distributions. Thus, there is strong potential for the genus to contain many more species, particularly in relatively poorly studied regions of eastern Africa including Zambia, Malawi and Tanzania.

This study investigates the diversity of the genus Zaireichthys by studying variation in the mitochondrial cytochrome oxidase subunit I (COI) barcoding gene. It focuses on species from five of African freshwater ecoregions: Lake Malawi, Mulanje, Middle Zambezi-Luangwa, Lake Rukwa and Coastal East Africa (Abell et al., 2008), and builds on results of a recent barcoding study of Zaireichthys from the Eastern Zimbabwe Highlands freshwater ecoregion (Chakona et al. 2018)

Hidden diversity of sand catlet fishes in the rivers of Eastern Africa August 2018

Materials and methods

Fishes were collected between May 2009 and August 2017, in Tanzania, Malawi and Zambia (Supporting Information Table 1). They were preserved in 70% ethanol. DNA was extracted from fins or tissues using the Blood and Tissue kit (Qiagen), and the presence of DNA was checked by using agarose gel electrophoresis. A fragment of the mitochondrial cytochrome oxidase subunit I (COI) gene was amplified by polymerase chain reaction (PCR) using universal fish DNAbarcoding primer set FishF1 and FishR1 (Ward et al., 2005). PCRs were performed with a Prime thermocycler (Techne). Each 25µL reaction mixture contained DNA template, 1µL of each primer, 12.5µL of “2x PCRBio Taq Mix Red” (PCRBiosystems) and 10.5µL of free-RNase water. The PCR amplification followed these steps: an initial denaturation at 94°C for 2 minutes, followed by 35 cycles of 95°C for 30 seconds, 55°C for 30 seconds and 72°C for 20 seconds, and then a final extension at 72°C for 6 minutes. PCR products were purified with Zymo Purification Kit (Zymo Research). PCR purified products were sequenced by Eurofins Genomics using ABI3730XL machines.

Our new sequences were aligned to previously published Zaireichthys sequences (Chakona et al. 2018) using the software DAMBE7 (Xia, 2018). The best-fitting model of nucleotide substitution was assessed using ModelFinder (Kalyaanamoorthy et al. 2017) implemented in the IQ-TREE software (Nguyen et al. 2015). The selection criterion was the corrected Akaike’s Information Criteria (AICc). Phylogenetic reconstructions were performed using BEAST2 (Bouckaert et al. 2014). Each analysis compromised 200 million generations, employing the Yule model and relaxed clock. Trees were saved every 1000 generations, and after removal of the first 25% of trees as “burn-in”, remaining trees then summarized onto a single consensus tree. The General Mixed Yule Coalescent (GMYC) approach (Pons et al. 2006; Fujisawa & Barraclough, 2013) was used to delineate potential species, performed with the “splits” package (Ezard & al., 2017) and R 3.5.1 (R Core Team, 2018). GMYC Results were compared to those obtained with multi-rate Poisson Tree Processes (mPTP; Kapli et al. 2017), performed on the mPTP server (https://mptp.h-its.org).

Results

The final alignment comprised 74 aligned sequences of 521 nucleotides were analysed, including 31 sequences generated for this study, 20 sequences from Chakona et al. (2018) and 23 sequences from the Barcode of Life database (http://v3.boldsystems.org/). In total, 31 unique haplotypes were identified. The GYMC approach identified 10 potential species, while the mPTP process was more conservative, and identified eight potential species (Figure 1). Of these eight species identified using the mPTP approach, which five were resolved within Chakona et al. (2018), while three are newly characterised in this study.

Within the newly sequenced specimens from Zambia, Malawi and Tanzania, we found representatives of four potential species (Figure 1). Representatives of Zaireichthys sp. “slender” (Chakona et al. 2018) were found in the Lake Malawi catchment (Bua River, Lutwembe River and Runyina River), in the eastward flowing Great Ruaha and Ruvuma catchments. Notably, the GMYC method delineated two potential lineages within Z. sp. ‘slender’, one distributed to the south and the east of Lake Malawi (including Ruvuma River system and Lower Zambezi system), and the second to the west and the north of the lake (including Lake Malawi, the Luangwa and Great Ruaha drainages).

Of the newly characterised species using the mPTP method, one Z. sp. “Igawa & Rukwa”, was widespread across the Lake Rukwa catchment, and also present within the neighbouring Great Ruaha River system. Hidden diversity of sand catlet fishes in the rivers of Eastern Africa August 2018

The GMYC method delineated representatives from the two catchments as distinct lineages. The second newly characterised species, Z. sp. ‘Ruaha’, only occurred in the Great Ruaha River. The third newly characterised species occurred in two rivers which flow into Lake Malawi (North Rukuru and Songwe rivers), and in Mlowo river which flows into Lake Rukwa.

Figure 1: Bayesian phylogenetic tree based on mitochondrial oxidase subunit I (COI) sequences. Node labels are posterior probabilities. The potential species were delineated with the GMYC method and the mPTP method. Hidden diversity of sand catlet fishes in the rivers of Eastern Africa August 2018

Figure 2: Distribution of Zaireichthys specimens in the region of interest. Orange spots represent Z. sp ‘slender’, red spots represent Z. sp. ‘Igawa & Rukwa’, purple spot represents Z. sp. ‘Ruaha’ and green spots represent Z. sp. ‘Malawi & Rukwa’. Hidden diversity of sand catlet fishes in the rivers of Eastern Africa August 2018

Discussion

Identity of delimited taxa. DNA barcoding has proven to be a convenient method of assessing genetic divergence among different populations of the same genus. Hence, it can be a powerful tool to guide and inform traditional taxonomic studies. Our analyses conservatively suggested the presence of eight Zaireichthys genetically delimited species within the dataset, of which two have been linked to morphologically formerly described species (Z. monomapata and Z. kavogoensis) from the Zambezi system. Three further previously recognised lineages taxa have not been formally linked to described species, including Z. sp. “Chilwa”, Z. sp “leopard spot” and Z. sp. “slender”. Our study adds a further three genetically recognised lineages, but we have not yet confidently assigned these to species. Several candidate species are known from the region we visited, including Z. compactus which is known from the Ruhuhu river or Tanzania, Z. maravensis from the rivers in the northwest of the Malawi catchment, Z. lacustris known from Lake Malawi itself, Z. rotundiceps known from central Tanzania and Z. wamiensis from central-east Tanzania (Eccles et al. 2011). The putative species delimited here using DNA barcode data require further investigation, from a detailed morphological point of view to, determine if they represent previously described species or novel undescribed taxa.

Phylogeography of Zaireichthys genus in Eastern Africa. Although the movement of freshwater species is restricted to connectivity within the river network, the distribution of putative species did not necessarily correspond with current patterns of river connectivity. There were several examples of the same candidate species being present in geographically separated water systems. For example, Z. sp. ‘Malawi and Rukwa’ was found in two different water systems, separated by a distance of 100 km and an altitude of 1000 m. According to mPTP method, Z. sp. ‘slender’ was widespread over more than 1000 km, in three different water systems. Zaireichthys is a fluvial species present in small streams, and plausibly is able to cross catchment boundaries during period flooding, or river capture events associated with geological activity. Additionally, we found potential species within the same water system. For example, three different candidate species (Z. sp. ‘slender’, Z. sp. ‘Igawa’ and Z. sp. ‘Ruaha’) were found in a small geographical area, in the Great Ruaha River system. Moreover, Z. sp. ‘slender’ and Z. sp. ‘Ruaha’ occurred in the same tributary of the Ruaha River and are still separated by 12.65% sequence divergence. Currently it is unknown if such populations are capable of hybridization, or whether consistence is facilitated by the use of different ecological niches along river systems, and within the same habitats.

DNA barcoding and phylogeny. The aim of this study is not to describe precisely new taxa, but to evaluate the diversity of Eastern African populations of Zaireichthys which, considering their relatively high genetic divergence, may represent new potential species. In light of the results of this study, it is quite probable that there is an underestimated diversity in this genus of African stream fishes. Moreover, there is a lack of knowledge on species distribution and vulnerability. Indeed, only 9 out 18 described species are listed in the IUCN red list database, and five are classified as “data deficient”. Since African freshwater habitats are directly threatened by human activities (agricultural expansion, mining, dam), and since Zaireichthys species often have restricted distributions, they could be particularly vulnerable. In addition, these might be endangered by the consequences of climate change. One species which occurred in Congo River bassin, Z. brevis, has already been identified as a species which is highly exposed and unable to adapt to climate change (Carr et al. 2013). Hidden diversity of sand catlet fishes in the rivers of Eastern Africa August 2018

Competing interest We have no competing interest.

Acknowledgments We thank the Zambia Department of Fisheries, Malawi Department of Fisheries and Tanzania Commission for Research and Technology (COSTECH) for permits. Fieldwork was supported by C. Katongo, H. Sungani, B. Ngatunga and A. Shechonge.

Funding Sample collection was funded by Royal Society-Leverhulme Trust Africa Awards AA100023 and AA130107 to MJG, while VV was supported during the work through an Erasmus scholarship and an University Paris- Saclay scholarship.

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