Skin coloration and habitat preference of the freshwater Anguilla eels

For centuries, the life history of freshwater eels (genus Anguilla) was a mystery to biologists. Freshwater eels are typical catadromous fi sh; they spend most of their lifetime in freshwater rivers and estuaries before fi nally migrating to their spawning grounds in the ocean for reproduction [1]. In total, there are 16 species and three subspecies of freshwater eels identifi ed in the world [2-4], which can be categorized into two groups based on body coloration: mottled skin and uniformly-colored skin, called “marbled eels” and “plain eels” [5]. Marbled eels are typically distributed mainly in tropical and subtropical regions, whereas plain eels are distributed over tropical, subtropical, and temperate regions [1]. Marbled and plain eels coexist in some rivers in tropical and subtropical regions, where they may share the same niches, and forage for the same prey, thereby causing possible interspecifi c competition [4]. Thus, under selection pressure, eels may evolve some morphological and physiological characters to reduce interspecifi c competition and maximize their sustainability. In recent years, several studies have been conducted on habitat choices of eels, and these revealed that they can be infl uenced by extrinsic environmental factors, such as salinity, water temperature, river size, and carrying capacity [6-8]. Other than these factors, the habitat preference of the eel in the river appears to be species-specifi c. For instance, the giant mottled eel (A. marmorata, a marbled eel) was inclined to inhabit the middle and upper reaches of rivers, but the Japanese eel (A. japonica, a plain eel) tended to dwell in the estuaries and lower reaches of rivers, as determined by analysis of trace elements (Sr/Ca ratios) in the otoliths taken from wild-captured eels in the same river [6,7,9], or by fi eld investigation [10]. Body coloration of animals facilitates a number of important physiological and behavioral functions, including thermoregulation, mate attraction, rival deterrence, and predator avoidance [11]. To better understand the life history strategy of the anguillid eels, especially the role of eel skin color on its habitat preference in the river, data were collected and analyzed to summarize the phylogenetic relationship, geographical distribution and habitat preference between marbled and plain eels. Abstract


Introduction
For centuries, the life history of freshwater eels (genus Anguilla) was a mystery to biologists. Freshwater eels are typical catadromous fi sh; they spend most of their lifetime in freshwater rivers and estuaries before fi nally migrating to their spawning grounds in the ocean for reproduction [1]. In total, there are 16 species and three subspecies of freshwater eels identifi ed in the world [2][3][4], which can be categorized into two groups based on body coloration: mottled skin and uniformly-colored skin, called "marbled eels" and "plain eels" [5]. Marbled eels are typically distributed mainly in tropical and subtropical regions, whereas plain eels are distributed over tropical, subtropical, and temperate regions [1]. Marbled and plain eels coexist in some rivers in tropical and subtropical regions, where they may share the same niches, and forage for the same prey, thereby causing possible interspecifi c competition [4]. Thus, under selection pressure, eels may evolve some morphological and physiological characters to reduce interspecifi c competition and maximize their sustainability.
In recent years, several studies have been conducted on habitat choices of eels, and these revealed that they can be infl uenced by extrinsic environmental factors, such as salinity, water temperature, river size, and carrying capacity [6][7][8]. Other than these factors, the habitat preference of the eel in the river appears to be species-specifi c. For instance, the giant mottled eel (A. marmorata, a marbled eel) was inclined to inhabit the middle and upper reaches of rivers, but the Japanese eel (A. japonica, a plain eel) tended to dwell in the estuaries and lower reaches of rivers, as determined by analysis of trace elements (Sr/Ca ratios) in the otoliths taken from wild-captured eels in the same river [6,7,9], or by fi eld investigation [10]. Body coloration of animals facilitates a number of important physiological and behavioral functions, including thermoregulation, mate attraction, rival deterrence, and predator avoidance [11]. To better understand the life history strategy of the anguillid eels, especially the role of eel skin color on its habitat preference in the river, data were collected and analyzed to summarize the phylogenetic relationship, geographical distribution and habitat preference between marbled and plain eels.
The habitats of freshwater eels in the river can be classifi ed into the upper reaches/mountain stream, middle reaches, and lower reaches/estuaries according to the hydrological conditions and the distance from the estuary. We searched online reference banks of Web of Science, Library of Congress, and Google Scholar till December 2019 to acquire habitat distribution information of anguillid eels. The search used combined key words like Anguilla, habitat, and distribution. We also searched specialist books and conference proceedings we had on hand regardless of the language. All acquired papers studying the biogeographic distribution and habitat preference of Anguilla eels were screened by manpower.

Evolutionary relationships of Anguilla eels
The evolutionary history of 19 Anguilla eels was inferred using the Neighbor-Joining method (Saitou & Nei, 1987) based on the complete mitochondrial genome ( Table 1). The marine eel Moringua edwardsi was used as the outgroup species. The optimal tree with the sum of branch length = 0.66905714 was shown. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) was shown next to the branches [12]. The evolutionary distances were computed using the Maximum Composite Likelihood method [13] in the units of the number of base substitutions per site. This analysis involved 20 nucleotide sequences. Codon positions included were 1st+2nd+3rd+Noncoding. All positions containing gaps and missing data were eliminated (complete deletion option). There were a total of 16350 positions in the fi nal dataset. Evolutionary analyses were conducted by MEGA X [14].

Relationship between body coloration and habitat usage of freshwater eels
With reference to previous studies, A. marmorata, A. luzonensis, A. bengalensis bengalensis, A. bengalensis labiata, A.
interioris, A. celebesensis, A. megastoma, and A. reinhardtii were classifi ed as marbled eels, and all other 11 species/subspecies were classifi ed as plain eels. The worldwide geographical distribution of marbled eels and plain eels showed that the marbled eels often overlapped with those of plain eels ( Figure   1) in tropical/subtropical areas.
Body coloration, geographical distribution, and habitat distribution data of all eel species were listed ( Table 2). A. marmorata was inclined to inhabit the upper reaches of rivers, while A. japonica, A. bicolor bicolor, and A. bicolor pacifi ca (all plain eels) tended to inhabit the lower reaches (brackish/sea waters) [6,7,10,15,16]. A. bengalensis bengalensis, the Indian mottled eel, was reported to reside from the upper-to midstream areas with cooler water temperatures and higher elevation [8,17]. The African mottled eel, A. bengalensis labiata, mainly inhabited areas far inland by surmounting formidable barriers in their upstream migration [18,19]. The Luzon mottled eel, A. in New Guinea [21], but the glass eels of A. interioris were ever detected at Java Island and southern Mindanao Island [22,23].
Another marbled eel, A. celebesensis, was reported to reside from the upper reaches of rivers in Sulawesi Island [24,25]. In addition to the Sulawesi Island, the glass eels of A. celebesensis were ever detected at Philippines coasts [26]. A. megastoma, the Pacifi c long-fi nned marbled eel, was found only in the middle or upper reaches near the headwaters of freshwater rivers [27,28]. The Australian long-fi nned marbled eel, A. reinhardtii, was one of main eel species at East Coast of Australia [29], and commonly found in many freshwater areas, including the upper reaches of rivers, dams, and lakes [30].
A. mossambica, a species of plain eel, is an inter-habitat migrant, moving between freshwater and seawater habitats, as indicated by microchemical analysis of their otoliths [31].
In another study, A. mossambica was found to inhabit coastal and estuarine areas without migration upstream into inland freshwater areas [32]. A. malgumora (A. borneensis), also called Indonesian longfi nned eel, was a common plain eel inhabiting the lower and middle reaches of rivers in Borneo and Sulawesi Island of Indonesia and Sabah of Malaysia [33,34]. A. obscura, the Pacifi c short-fi nned plain eel, was distributed primarily over the lower reaches of rivers and coastal lagoons at western South Pacifi c, including Papua New Guinea, Fiji, and French Polynesia [27,35,36]. The other two short-fi nned plain eels,

Phylogeny analysis of Anguilla eels in relationship to skin color and habitat type
The phylogenetic relationships, skin color and temporal/ tropical characters of 19 Anguilla eels were constructed in Figure 2. Generally, eels with closer phylogenetic relationships possess similar morphological and distribution characteristics.
Most marbled eels shared a close phylogenetic relationship, and, likewise, most of the plain eels shared a close phylogenetic relationship. All marbled eels distribute in tropical/subtropical areas, and all temporal eels have plain skin colors.

Discussion
Teleost fi sh develop at least six classes of pigment cells (chromatophores) leading to a fascinating diversity of colors [41]. anguillid eels are characterized with a dark or marbled back produced by black/brown melanophores and yellow/ orange xanthophores at the inner and outer boundaries of the dermis [42]. The mechanism in controlling skin color pattern of the anguillid eels may be related to Agouti signaling protein [41]. Some animals adapt to their environment through body coloration. For example, chameleons maintained crypsis and communicated by changing their body coloration [43].
Cephalopods, such as octopuses, can rapidly change their body coloration to mimic visual characteristics of the background [44]. Another method of camoufl age, known as countershading, has been reported in fi sh species [11,45]. Some body coloration of fi sh, including eels, is darker on the upper side and brighter on the underside. When seen from the underside, the brighter ventral body would blend in with the light background. In contrast, when seen from the top in daylight, the darker dorsal surface would blend in with the dark background of the deep water or substrate below [46]. Freshwater eels may also have evolved different body colorations (marbled and plain) in order to blend in with their habitat.
The comprehensive analysis of this study indicated that, in tropical/subtropical areas where marbled and plain eels coexist, marbled eels tended to inhabit the middle to upper reaches of rivers while plain eels prefer the habitats located from the middle reaches to estuaries (Table 2). In a typical river system of tropical/subtropical areas, the substrates found in upper reaches are primarily boulders, bedrock, cobble, and gravel, which are quite different from the substrates of the lower reaches, which are more sandy and muddy because rivers deposit eroded materials carried from the upper reaches [47].   reaches, and A. megastoma (marbled) using the upper reaches in rivers of French Polynesia [27]. The potential for interspecifi c competition between eel species in these rivers could be effectively reduced by this habitat partitioning strategy. Interestingly, recent study indicated that hybridization and gene exchange are pervasive in tropical eel species, but that these species have nevertheless remained distinct entities for several million years. Cytonuclear incompatibilities and hybrid breakdown may be powerful mechanisms which help maintaining species boundaries [49].
Phylogenetic tree revealed that most marbled eels shared a close phylogenetic relationship, and, likewise, most of the plain eels shared a close phylogenetic relationship ( Figure  2). The eels with closer phylogenetic relationships possessed similar skin coloration, which probably results from their long-term adaptive evolution. It is also worth noting that only plain eels inhabited the temperate areas. In temperate areas, the upper reaches of rivers are usually characterized by low primary productivity and low temperature, thus lacking suitable habitats for eels. Subsequently, eels usually inhabit the estuaries and lower reaches of rivers in temperate areas. Marbled eels which have evolved to adapt tropical/subtropical areas were thus rare in temperate area.
Previous study indicated that American eels could be distributed over the middle reaches of rivers in temperate areas [39]. A recent study has also revealed that the giant mottled eel is distributed throughout estuaries and the middle and upper reaches of some short tropical rivers [50][51][52][53][54]. These large-scale habitat distributions may result from a lack of interspecifi c competition. That is, eels may be able to expand their habitat range to some extent if no competitive species exist. Even so, the habitat partitioning between plain and marbled eels in the same river is a common phenomenon in almost all geographical areas.
The differences in body coloration between marbled eels and plain eels are likely a result of adaptive evolution which may help to reduce potential interspecifi c competition when they coexist in the tropical/subtropical rivers. This fi nding is important for developing applicable management plans for eels with different skin color types, with lower reach/estuary for protecting plain eels and middle/upper reaches for conserving marbled eel species.