Enteromius Cope, 1867

Ginneken, Marjolein Van, Decru, Eva, Verheyen, Erik & Snoeks, Jos, 2017, Morphometry and DNA barcoding reveal cryptic diversity in the genus Enteromius (Cypriniformes: Cyprinidae) from the Congo basin, Africa, European Journal of Taxonomy 310, pp. 1-32 : 7-12

Genus Enteromius Cope, 1867

Evaluation of the literature-based identifications

In the ML tree obtained ( Fig. 2 ), lineages with less than 2% sequence divergence were collapsed and named after the river system from which the specimens were collected. Specimens from the Lomami/ Lobaye system and the Lobilo were grouped under the label ‘Kisangani region’, since there appeared to be little or no genetic difference between samples of these nearby affluents.

The ML tree shows 23 lineages within the assayed Enteromius samples, representing the four ‘a priori’ morphospecies in the following quantities/properties: 13 of which belong to the E. cf. miolepis group; three to the E. cf. brazzai group; one to the E. cf. pellegrini group; and six to the E. cf. atromaculatus group. Enteromius cf. miolepis , E. cf. atromaculatus and E. cf. pellegrini each form clearly supported clades, while this is not the case for E. cf. brazzai . There is often a substantial genetic distance between lineages occurring in different rivers, but also between some lineages detected in the same river. For example, within the E. cf. miolepis group, we observed a considerable difference between specimens of the Luapula (three lineages; 14.6% sequence divergence between Luapula 1 and 2; 15.3% between Luapula 1 and 3; 19.0% between Luapula 2 and 3),and the Luki (two lineages; 7.7% sequence divergence). Interestingly, the sequences obtained for the Upper, Middle and Lower Congo populations did not group according to these zoogeographic regions. However, two out of the three lineages of the Luapula, a river system geographically remote from the other rivers included in this study, also show the largest genetic distances within the E. cf. miolepis clade. Enteromius cf. brazzai is not resolved as a single clade, and consists of three lineages, two of which were detected in the ‘Kisangani region’ (19.8% sequence divergence). In contrast to the other groups, E. cf. pellegrini consists of a single lineage that is composed of specimens from a single river, the Ituri. The E. cf. atromaculatus group contains an important amount of genetic variation, with five of its six lineages occurring in the Ituri River. Remarkably, one of those lineages also contains one sample from the ‘Kisangani region’.

Morphometric groupings

Firstly, PCAs were executed on all the specimens examined, after which more detailed PCAs were performed on each literature-based ‘a priori’ group separately.

Overall analyses

The highest loadings on PC2 for a PCA on 17 log-transformed measurements (n = 177) are for the eye diameter (ED), the post-anal distance (PoAD) and the dorsal fin length (DoFL); PC1 is a proxy for size (see above). On a scatterplot of PC2 against PC1, the four ‘a priori’ groups cannot all be distinguished from each other ( Fig. 3 ). However, specimens of E. cf. pellegrini and E. cf. brazzai are completely separated from each other on PC2. This is mainly because E. cf. pellegrini has a smaller ED, a smaller dorsal fin base length (DoFBL) and a smaller PoAD than E. cf. brazzai . Clearly E. cf. miolepis occupies the largest morphospace on PC2, which comprises most of the type specimens included, except for two paralectotypes of E. eutaenia and one syntype of E. holotaenia . The E. cf. atromaculatus polygon comprises the paratypes of E. atromaculatus . The specimens of E. cf. pellegrini , however, only overlap slightly with the type specimens of E. pellegrini and the specimens of E. cf. brazzai are separated from the holotype of E. brazzai and types of E. tshopoensis . Furthermore, the type specimens of E. tshopoensis are separated from the other groups, mainly on PC2, due to a smaller PoAD and a larger DoFL.

The highest loadings on PC1 for a PCA on 10 meristics (n = 177) are for the number of scales between the lateral line and the belly (L-B Sc), the number of scales between the dorsal fin and the lateral line (D-L Sc) and the number of scales between the lateral line and the pelvic fin (L-P Sc); on PC2 for the number of scales on the lateral line (LL Sc), the number of scales between the occiput and the base of the first dorsal fin ray (PD Sc) and the number of pelvic fin rays (PelFR). Similar to the analysis for the measurements, a scatterplot of PC2 against PC1 does not allow the separation of the four ‘a priori’ groups ( Fig. 4 ). Again, E. cf. miolepis is the group showing the largest variation. Also, E. cf. pellegrini and E. cf. brazzai are again completely separated on PC1, mainly due to E. cf. pellegrini having a higher D-L Sc (4.5–5.5 vs 3.5) and a higher L-B Sc (5–6 vs 4–5). Furthermore, the four ‘a priori’ groups all overlap with their respective type specimens.

To examine whether morphological differences could be detected between the different genetic lineages within each group, we performed PCAs on E. cf. miolepis , E. cf. brazzai and E. cf. atromaculatus separately. To investigate whether one or more lineages represent a currently valid species, relevant type

specimens were also included in these analyses. This resulted in a multitude of analyses for which the most important outcomes are presented below.

Morphometric comparisons among E. cf. miolepis lineages

Because of the high number of lineages and specimens for E. cf. miolepis , we analysed the major geographical regions, the Upper, the Middle and the Lower Congo, separately (see also Table 1 ), but still included the relevant type specimens to check whether some groups could be allocated to these species. Only the results of the latter are discussed as an example.

For the Lower Congo, we detected three genetic lineages within E. cf. miolepis ( Fig. 2 ), i.e., one group containing specimens from the Inkisi and two groups with specimens from the Luki (Luki 1 and Luki 2). The highest loadings on PC1 for a PCA on 10 meristics (n = 36) are for LL Sc, CP Sc and PecFR; on PC2 again for PecFR, AFR and PD Sc. The specimens from the Inkisi are well separated from the Luki lineages on both PC1 and PC2; and there is only one specimen overlap between Luki 1 and Luki 2 ( Fig. 5 ). In addition, specimens from these latter two lineages noticeably differ in the length of their barbels. Lineages Inkisi and Luki 1 can be clearly distinguished from all type specimens ( Fig. 5 ). Luki 2 overlaps with the type specimens of E. eutaenia and E. holotaenia , but Luki 2 differs from the E. eutaenia types in barbel length. A PCA of the log-transformed measurements did not separate the genetic lineages from each other or from the type specimens (not illustrated).

For the Middle Congo and Upper Congo, we detected respectively six and four genetic lineages of E. cf. miolepis ( Fig. 2 ). Similar exploratory morphometric analyses of these genetic lineages, as for the Lower Congo discussed above, indicated that almost all could be separated from each other as well as from the included type specimens. However, specimens from ‘Kisangani region’ 1, Itimbiri and the syntypes of E. holotaenia clustered on the PCAs. Yet, the Itimbiri specimens had a different colour pattern (a black anal fin tip), which is absent in the ‘Kisangani region’ 1 specimens as well as in the syntypes of E. holotaenia .

When comparing all lineages of E. cf. miolepis (Lower, Middle and Upper Congo), each could be morphologically distinguished from the others based on meristics ( Fig. 6 ), measurements and/or barbel length (not illustrated). Only specimens from Kisangani region’ 1 and Itimbiri could not be separated from each other, but differed in colour pattern (see above).

Morphometric comparisons among E. cf. brazzai lineages

We observed three genetic lineages within E. cf. brazzai ( Fig. 2 ). The highest loadings on PC1 for a PCA on 10 meristics (n = 22) are for LL Sc, PecFR and PelFR; on PC2 for CP Sc, L-B Sc and again PecFR. Specimens from the ‘Kisangani region’ 2 are well separated from the Ituri 3 lineage on PC2 and both genetic lineages differ from the ‘Kisangani region’ 3 lineage and type specimens on PC1 ( Fig. 7 ). The polygon of specimens from the ‘Kisangani region’ 3 lineage comprises the holotype of E. brazzai and overlaps with the types of E. tshopoensis . However, the eight specimens from the ‘Kisangani region’ 3 lineage as well as the holotype of E. brazzai differ from the types of E. tshopoensis by the absence of barbels.

Morphometric comparisons among E. cf. atromaculatus lineages

We detected six genetic lineages within E. cf. atromaculatus ( Fig. 2 ). Although there was only 1.75% sequence divergence between the lineages of Epulu 2 and Ituri 8, we interpreted these lineages as separate Operational Taxonomic Units (OTUs, i.e., clusters of similar DNA sequences) because of the observed differences in colour pattern (specimens from the Epulu 2 lineage have mid-lateral dots, while specimens from Ituri 8 display a vague mid-lateral band). The single specimen from Ituri 7 was lost and could not be measured. The highest loadings on PC1 for a PCA of 10 meristics (n = 42) are for LL Sc, PecFR and D-L Sc; on PC2 for CP Sc, again PecFR and D-L Sc. A plot of PC2 vs PC1, separates the two specimens from the Ituri 6 lineage from all other lineages based on PC2 ( Fig. 8 ). The specimens from the Ituri/‘Kisangani region’ lineage are separated from all other lineages mainly on PC1. The Ituri 5 lineage is separated from the Ituri 6 and Epulu 2 lineages along PC2, from the E. atromaculatus type specimens on PC1, and from the Ituri/’Kisangani region’ and Ituri 8 lineages on a combination of PC1 and PC2.

As the initial PCA resulted in a great overlap between the Epulu 2 and Ituri 8 lineages, we carried out a second PCA on 10 meristics, only including specimens from these lineages and the types of E. atromaculatus . The highest loadings on PC1 are for LL Sc, D-L Sc and CP Sc; on PC2 for PecFR, PD Sc and the number of pelvic fin rays (PelFR). On a plot of PC2 versus PC1, specimens from Epulu 2 and Ituri 8 still overlap and the types of E. atromaculatus overlap with specimens from Epulu 2 ( Fig. 9 ). These groups also overlap on a PCA of 17 log-transformed measurements (not illustrated).

Figures

Fig. 2. A. ML tree based on 558-bp-long Enteromius COI sequences with 1000 bootstrap replications, with node support shown as NJ/ML bootstrap (bootstrap values> 95% are shown; lineages <2% sequence divergence were collapsed), the label ‘Kisangani region’ contains samples from the Lomami/ Lobaye system and the Lobilo. B. Map of the Congo basin with the sampled river stretches indicated according to the phylogenetic lineages.

Fig. 3. Scatterplot of PC2 against PC1 for a PCA on 17 log-transformed measurements (n = 177) of Enteromius Cope, 1867: E. cf. miolepis (Boulenger, 1902) (◊), E. cf. brazzai (Pellegrin, 1901) (♦), E. cf. pellegrini (Poll, 1939) (∆), and E. cf. atromaculatus (Nichols & Griscom, 1917) (▲). Also shown are the type specimens examined of: E. miolepis (Boulenger, 1902) (○), E. holotaenia (Boulenger, 1904) (●), E. eutaenia (Boulenger, 1904) (□), E. kerstenii (Peters, 1868) (■), E. brazzai (Pellegrin, 1901) (), E. tshopoensis (De Vos, 1991) (▼), E. pellegrini (Poll, 1939) (+), and E. atromaculatus (Nichols & Griscom, 1917) ().

Fig. 4. Scatterplot of PC2 against PC1 for a PCA on 10 meristics (n = 177) of Enteromius: E. cf. miolepis (Boulenger, 1902) (◊), E. cf. brazzai (Pellegrin, 1901) (♦), E. cf. pellegrini (Poll, 1939) (∆), and E. cf. atromaculatus (Nichols & Griscom, 1917) (▲). Also shown are the type specimens examined of: E. miolepis (Boulenger, 1902) (○), E. holotaenia (Boulenger, 1904) (●), E. eutaenia (Boulenger, 1904) (□), E. kerstenii (Peters, 1868) (■), E. brazzai (Pellegrin, 1901) (), E. tshopoensis (De Vos, 1991) (▼), E. pellegrini (Poll, 1939) (+), and E. atromaculatus (Nichols & Griscom, 1917) ().

Fig. 5. Scatterplot of PC2 against PC1 for a PCA on 10 meristics (n = 36) of E. cf. miolepis specimens from the Lower Congo: Inkisi (◊), Luki 1 (♦) and Luki 2 (∆). Also shown are the type specimens examined of: E. miolepis (Boulenger, 1902) (○), E. holotaenia (Boulenger, 1904) (●), E. eutaenia (Boulenger, 1904) (□) and E. kerstenii (Peters, 1868) (■).

Fig. 6. Scatterplot of PC2 against PC1 for a PCA on 8 meristics (n = 60) of E. cf. miolepis (Boulenger, 1902) specimens from the Congo basin (excluding types): ‘Kisangani region’ 1 (◊), Ituri 1 (♦), Itimbiri (∆), Léfini (▲), Epulu 1 (○), Inkisi (●), Luapula 1 (□), Luki 1 (■), Luapula 2 (), Luapula 3 (▼), Ituri 2 (+), and Luki 2 (). Specimens from Luapula 1 and Luapula 2 can be separated from each other based on a PCA on the log-transformed measurements; specimens of Luki 2 fall separated when barbel lengths are included; specimens from Kisangani region’ 1 and Itimbiri can be distinguished based on colour pattern.

Fig. 7. Scatterplot of PC2 against PC1 for a PCA on 10 meristics (n = 22) of E. cf. brazzai (Pellegrin, 1901): ‘Kisangani region’ 2 (◊), Ituri 3 (♦) and ‘Kisangani region’ 3 (∆). Also shown are the type specimens examined of E. brazzai (Pellegrin, 1901) (○) and E. tshopoensis (De Vos, 1991) (●).

Fig. 8. Scatterplot of PC2 against PC1 for a PCA on 10 meristics (n = 42) of E. cf. atromaculatus (Nichols & Griscom, 1917): Ituri 5 (◊), Ituri 6 (♦), Ituri/‘Kisangani region’ (∆), Epulu 2 (▲), and Ituri 8 (). Also shown are the type specimens of E. atromaculatus (Nichols & Griscom, 1917) (○).

Fig. 9. Scatterplot of PC2 against PC1 for a PCA on 10 meristics (n = 36) of E. cf. atromaculatus (Nichols & Griscom, 1917): Epulu 2 (▲), and Ituri 8 (). Also shown are the type specimens of E. atromaculatus (Nichols & Griscom, 1917) (○).