Journal of Threatened
Taxa | www.threatenedtaxa.org | 26 September 2023 | 15(9): 23827–23835
ISSN 0974-7907
(Online) | ISSN 0974-7893 (Print)
https://doi.org/10.11609/jott.8491.15.9.23827-23835
#8491 | Received 26
April 2023 | Final received 07 September 2023 | Finally accepted 11 September
2023
Phylogenetic insights on the
delineation of Mysore and Malabar subspecies of the Grey Slender Loris Loris lydekkerianus in
southern India
Vinay Teja
1, Shivakumara Manu 2, Honnavalli N.
Kumara 3 &
Govindhaswamy Umapathy
4
1,2,4 CSIR-Centre for Cellular and
Molecular Biology, Uppal Road, Habsiguda, Hyderabad,
Telangana 500007, India.
3 Salim Ali Centre for Ornithology
and Natural History, Anaikatty (POST), Coimbatore,
Tamil Nadu 641108, India.
1 vinay@ccmb.res.in, 2 smanu@ccmb.res.in,
3 honnavallik@gmail.com, 4 guma@ccmb.res.in
(corresponding author)
1,2 contributed equally.
Editor: K.A.I. Nekaris,
Oxford Brookes University, Oxford, United Kingdom. Date of publication: 26 September 2023
(online & print)
Citation: Teja, V., S. Manu, H.N. Kumara
& G. Umapathy (2023). Phylogenetic
insights on the delineation of Mysore and Malabar subspecies of the Grey
Slender Loris Loris lydekkerianus
in southern India. Journal
of Threatened Taxa 15(9): 23827–23835. https://doi.org/10.11609/jott.8491.15.9.23827-23835
Copyright: © Teja et al. 2023. Creative Commons Attribution 4.0
International License. JoTT allows unrestricted use, reproduction, and
distribution of this article in any medium by providing adequate credit to the
author(s) and the source of publication.
Funding: Council
of Scientific and Industrial Research (CSIR) and Department of Biotechnology (DBT), Govt. of India.
Competing interests: The authors declare no competing interests.
Author details: Vinay Teja is a PhD student at CSIR-CCMB. He is interested in Bioinformatics and Molecular evolution of traits in primates.
Shivakumara Manu is a PhD student at
CSIR-CCMB. He holds a Master’s degree in Bioinformatics and Applied Biotechnology and is interested in solving biological problems through genomics and computational approaches.
Dr. Honnavalli N. Kumara is a wildlife biologist working as a Principal Scientist at SACON. His interest lies in understanding population dynamics, behavioural ecology, and conservation of primates, other mammals and birds.
Dr. Govindhaswamy Umapathy is a conservation biologist working as a Senior Principal Scientist and Group Leader at the LaCONES, CSIR-CCMB. His research interests include understanding species extinction in human dominated landscapes, genome sequencing and developing biotechnological tools for biodiversity conservation.
Author contributions: G.U. and H.N.K conceived the idea for this study and collected the samples. V.T. and S.M. analyzed the data and drafted the manuscript. All authors contributed to the revision of the manuscript and agreed to the final version of the manuscript.
Acknowledgements: V.T. was supported by a CSIR PhD
fellowship, S.M. was supported by a DBT-BINC PhD fellowship, and G.U. was
supported by CSIR, Govt. of India. We thank Dr. Mihir
Trivedi for providing useful suggestions for the phylogenetic analysis.
Abstract: Slender lorises are a threatened
genus of small and nocturnal strepsirrhine primates confined to India and Sri
Lanka. The Grey Slender Loris Loris lydekkerianus is divided into several subspecies based
on morphological variation and geographical distribution but not supported by
molecular data. We investigated the phylogenetic divergence of two subspecies
of the Grey Slender Loris in southern India: the Mysore Slender Loris Loris lydekkerianus ssp.
lydekkerianus and the Malabar Slender Loris Loris lydekkerianus ssp.
malabaricus. We generated whole genome shotgun
sequence data and assembled the whole mitochondrial genomes of representative
individuals from their distribution in southern India and compared them with
publicly available mitogenomes of other lorises. We found that the Mysore and
Malabar Slender Lorises vary by 2.09% in the COX1 and CYTB gene regions.
Further, phylogenetic analysis of 13 protein-coding and two ribosomal RNA genes
in the mitochondrial genome showed that the Mysore and Malabar Slender Lorises
form distinct monophyletic clades that diverged about 1.049 million years ago,
shortly after the divergence of Red Slender Loris Loris
tardigradus. Considering this relatively high
sequence variation and evolutionary divergence together with their already
established morphological differences and geographically distinct habitats, we
propose to recognize the Mysore and Malabar Slender Lorises as two distinct
species Loris lydekkerianus and Loris malabaricus.
Keywords: Grey Slender Loris, Malabar
Slender Loris, molecular dating, Mysore Slender Loris, phylogenetics.
Introduction
Slender lorises (genus Loris)
are one of the two genera of extremely specialized nocturnal primates that
inhabit India (Nekaris 2014). They belong to the
family Lorisidae, which also includes Slow lorises, Pottos, and Angwantibos. Slender lorises are confined to
India and Sri Lanka, where they inhabit dry to moist and lowland to montane
forests (Singh et al. 2021). Slender lorises are characterized by their small
size, long limbs, vestigial tail, large eyes, and slow locomotion. They are
adapted for arboreal life, using their opposable thumbs and toes to grasp
branches and their binocular vision to judge distances, visual acuity, precise
hand-eye coordination and social communication. They feed mainly on insects but
also consume fruits, flowers, gums, and other plant materials (Nekaris & Rasmussen 2003; Radhakrishna & Kumara 2010). They have a variety of vocalizations that may
help them avoid predators and communicate with conspecifics (Radhakrishna &
Singh 2002). Slender lorises are divided into two species: the
Grey Slender Loris Loris lydekkerianus
found in southern India and Sri Lanka and the Red Slender Loris Loris tardigradus
found only in Sri Lanka (Groves 2001). Both species show high phenotypic
variation in fur color, body size, and cranial morphology, leading to the
recognition of several subspecies, most of which are refuted by molecular
studies (Nijman et al. 2020). The Mysore Slender Loris Loris
lydekkerianus ssp. lydekkerianus
(Image 1) and the Malabar Slender Loris Loris
lydekkerianus ssp. malabaricus
(Image 2), which live in the dry and wet forests of the Eastern and Western
Ghats, respectively, are the two subspecies that have been recognized thus far
in southern India (Kumara et al. 2013). There are
several regions in their distribution where slender lorises face serious threats
to their existence such as habitat loss due to deforestation and urbanization,
electrocution on live wires, road accidents, pet trade, and illegal poaching
for traditional medicine and black magic (Dittus et
al. 2022; Gnanaolivu et al. 2022). The IUCN Red List
of Threatened Species classifies the Mysore Slender Loris (Kumara
et al. 2022a) and Malabar Slender Loris (Kumara et
al. 2022b) as ‘Near Threatened’ and they are listed under Schedule I of the
Indian Wild Life (Protection) Act, 1972. Recently, Tamil Nadu became the first
Indian state to notify a sanctuary for slender lorises spanning 118.06 km2,
which is crucial for protecting their habitat and ensuring the survival of this
unique primate species (Government of Tamil Nadu 2022).
The Mysore and Malabar subspecies
of the Grey Slender Loris were delineated based on their morphological
differences and geographic distribution (Groves 2001; Kumara
et al. 2013). The Mysore Slender Loris is relatively larger (ca. 260 g) than
Malabar Slender Loris (ca. 180 g) (Kumara et al.
2006). The Mysore Slender Loris has a grayish-brown coat and a prominent white
stripe on its forehead, whereas the Malabar Slender Loris has a reddish-brown
coat and a less distinct forehead stripe (Groves 2001; Kumara
et al. 2006). The relative distribution of the two subspecies as well as their
comparative densities and the extent of overlap between their distributions
have been very well established (Kumara et al. 2013).
The Mysore Slender Loris is found in the Eastern Ghats and eastern foothills of
the southern Western Ghats, while the Malabar Slender Loris is confined to the
western slope of the entire Western Ghats (Kumara et
al. 2013). The Mysore Slender Loris prefers dry deciduous forests with moderate
canopy cover and high tree density, while the Malabar Slender Loris prefers
moist evergreen forests with high canopy cover and low tree density (Kumara et al. 2013). Their distributions overlap along the
southern ridges of the Western Ghats, where hybridization may occur. While the
diet of Mysore Slender Loris mostly consists of insects, plant material, and
gum, the feeding behavior of the Malabar Slender Loris is not well studied
(Radhakrishna & Kumara 2010). The reproductive
biology and social system of the Mysore subspecies is influenced by factors
such as seasonality, food availability, predation risk, and population density.
It also has a seasonal breeding cycle that coincides with periods of high food
availability (Radhakrishna & Singh 2004). No such information on the
reproductive biology of the Malabar subspecies is available. Behavioral studies
on lorises have always been more challenging than on relatively large, diurnal,
and group-living primates such as macaques and langurs because they are
nocturnal, small in size, and mostly semi-gregarious. Given the distinct
habitat preferences and morphology of these two subspecies, understanding their
evolutionary history and genetic differences is vital to address their
conservation status and management issues.
Therefore, the main objective of
this study is to investigate the phylogenetic relationship and genetic
divergence between the Mysore and Malabar Slender Lorises in southern India. To
achieve this, we sequenced and assembled the whole mitochondrial sequences from
three representative samples. We aligned these sequences with the publicly
available sequences of other lorises and constructed phylogenetic trees. We
estimated the sequence divergence and divergence time between the two
subspecies using phylogenetic analysis. Our results support the morphological
and geographical delineation of the Malabar and Mysore Slender Lorises and
advocates for recognizing them as two distinct species. This study will
contribute to the understanding of the biogeography and speciation processes of
these threatened lorises and provide crucial insights for their conservation
and management.
Materials
and Methods
Sample Collection, DNA
extraction, and Sequencing
We followed the sample collection
guidelines of the animal ethics committees of the CSIR-Centre for Cellular and
Molecular Biology and Salim Ali Centre for Ornithology and Natural History.
Necessary permissions for sample collection were obtained from the Central Zoo
Authority of India, Ministry of Environment, Forests & Climate Change,
Government of India, vide Ref. No. 9-2/2005-CZA(M) Vol III. Rescued lorises of
known wild origin within the IUCN designated ranges (Figure 1) that were
captive in Mysore and Hyderabad zoos were the sources of our samples. Blood
samples were collected in EDTA vacutainers by qualified zoo veterinarians from
three representative individuals of Loris lydekkerianus
ssp. lydekkerianus (N = 2) and Loris lydekkerianus ssp. malabaricus
(N = 1). We used the Qiagen DNeasy Blood and
Tissue Kit to isolate the genomic DNA from the blood samples. We measured the
quality and quantity of genomic DNA using Nanodrop and Qubit 4. We constructed
whole genome libraries using the Truseq PCR-free
library preparation kit according to Illumina’s protocols. Briefly, 1 ug of genomic DNA was sheared to approximately 350 bp using the Covaris ultrasonicator. The fragmented DNA was then end-repaired
and blunt-end ligated with sequencing adapters containing unique dual indices
from IDT. The library was then size-selected using SPRI beads and verified on
the Agilent fragment analyzer. The cleaned-up libraries were finally quantified
in qPCR using the standards and Illumina adapter-specific primers from the
Roche library quantification kit. Libraries having good concentration were pooled
along with other samples and sequenced on the Illumina Novaseq
6000 platform for 300 cycles in paired-end mode.
Mitochondrial genome assembly
We demultiplexed the base call
files to separate the three samples with the dual-indexed barcodes using the BCL2FASTQ
tool from Illumina. Raw reads were quality-filtered with a phred
quality score threshold of 15 using FASTP v0.20 (Chen et al. 2018). We
subsampled 10 million quality filtered reads to de novo assemble the circular
mitochondrial genomes of all three samples using GetOrganelle
v1.7.1 (Jin et al. 2020). We then annotated all the
mitogenomes using MITOS2 (Bernt et al. 2013) with the
Refseq 89 Metazoa reference
mitochondrial database and the vertebrate mitochondrial genetic code. All the
coding and non-coding genes were extracted from the mitochondrial genomes using
the annotations.
Sequence and Phylogenetic
analyses
We aligned the full-length COX1
and CYTB genes of lorises using Clustal Omega with
the “distmat” flag and calculated the pairwise distances
between the sequences (Sievers & Higgins 2021). To build the phylogeny,
along with our samples we used the NCBI RefSeq
mitochondrial sequences from strepsirrhines namely, Loris lydekkerianus, Loris tardigradus,
Nycticebus coucang,
Nycticebus bengalensis,
Galago senegalensis, and Lemur catta.
We aligned the 13 protein-coding genes and two non-coding ribosomal RNA genes
individually from the assembled mitochondrial genomes and reference sequences
using the MUSCLE algorithm in MEGA7 (Kumar et al. 2016) and checked for the
presence of any sequencing errors or frameshifts for codon position. We then
concatenated all the gene alignments using MEGA7 (Kumar et al. 2016) and
identified the optimum nucleotide substitution model for each partition based
on the corrected Akaike information criterion (AICc)
values using PartitionFinder2 (Lanfear et al. 2017)
(Supplementary file 1). We built the maximum likelihood (ML) tree based using
IQ-TREE (Minh et al. 2020) with 1000 times bootstrapping. The ML tree was
visualized in Evolview v3 (Subramanian et al. 2019).
We utilized BEAST2.5 (Bouckaert et al. 2014) to create a divergence time tree
using the same concatenated alignment of 13 coding and two non-coding genes
from the complete mitochondrial genomes. We used the same partitioning scheme
and substitution models identified by PartitionFinder2. We then chose two
fossil calibration points:
1) We calibrated the crown node
of Galagos with 38 mya
based on the age of the fossil Saharagalago
misrensis (PaleoDB
collection 67706) (Seiffert et al. 2003). We applied
a normal distribution at 40 Mya (SD = 0.04; 95% range: 36–43)
2) We calibrated the crown node
of Slow Lorises with 13.82 mya based on the age of
the fossil Nycticebus linglom
(PaleoDB collection 48126) (Harrison 2010). We
applied a normal distribution at 14 Mya (SD = 0.05; 95% range: 9–17)
For all the partitions, we
created a relaxed lognormal clock and employed a birth-death process using
prior distributions. To get to the final tree, we ran for 40 million generation
runs, sampling every 2,000th generation using TreeAnnotator
(Helfrich et al. 2018) with a 10% burn-in. We verified that all the ESS values
were over 200 in Tracer 1.7 (Rambaut et al. 2018) and
visualized the tree in FigTree v1.4.4 (Rambaut 2014).
Results
Phylogenetic analyses support the
morphological and geographical delineation of Mysore & Malabar Slender
Lorises
To investigate the genetic
differences between the two subspecies of slender loris in southern India, we
first assembled three new circular mitochondrial genomes with an average length
of 16,771 bp from two samples of the Mysore Slender
Loris Loris lydekkerianus
ssp. lydekkerianus and one sample of
Malabar Slender Loris Loris lydekkerianus ssp. malabaricus.
We annotated the mitochondrial genomes along with published reference sequences
and obtained the full-length sequences of 13 protein-coding genes and two
ribosomal RNA genes. To check the variation in the nucleotide sequence within
the Loris genus, we estimated the pairwise sequence similarity in the
COX1 and CYTB regions spanning 2,682 bp between all
six Loris samples (Table 1). We observed the highest average sequence variation
of 2.82% (S.D. 0.16) between the four sequences of the Grey Slender Loris Loris lydekkerianus
and the two sequences of the Red Slender Loris Loris
tardigradus as they belong to two different
species within the Loris genus. While there was no sequence variation
found within the two sequences of Red Slender Loris, there was considerable
variation within the four sequences of Grey Slender Loris contributed by the
difference between the two subspecies. We found about 2.09% (S.D 0.0) variation
in the COX1 and CYTB sequences of the Mysore and Malabar Slender Lorises.
We then used phylogenetic
analyses to understand the evolutionary relationships between the two
subspecies. Along with our three samples, we included reference mitochondrial
sequences from two species of slender lorises (L. lydekkerianus,
L. tardigradus) and two species of slow
lorises (Nycticebus bengalensis,
N. coucang) along with galago and lemur
as outgroups (Figure 2). The phylogenetic tree recapitulates the broad
evolutionary relationships of slender lorises with slow lorises and the
outgroups. It reveals an interesting pattern within the clade of slender lorises
where the Mysore Slender Loris L.l.
ssp. lydekkerianus clusters with the reference
sequence of Grey Slender Loris to form a monophyletic clade and the Malabar
Slender Loris L.l. ssp. malabaricus forms a separate monophyletic clade with
very strong statistical support. We noted that the Malabar Slender Loris
appears more closely related to the Red Slender Loris L. tardigradus, albeit with a very small branch length
(Figure 2). To estimate the divergence time between the two subspecies and
other lorises, we constructed a fossil-calibrated Bayesian tree (Figure 3). Our
results suggest that the split between the Grey Slender Loris L. lydekkerianus and Red Slender Loris L. tardigradus occurred approximately 1.087 million
years ago (mya). This was immediately followed by
diversification of the Mysore Slender Loris L.l.
ssp. lydekkerianus
and Malabar Slender Loris L.l. ssp. malabaricus at
around 1.049 mya (Posterior probability = 1) (Figure
3).
Discussion
Our results from the phylogenetic
analyses based on the mitochondrial sequences show that the Mysore and Malabar
Slender Lorises have significant genetic variation (2.09%) in the COX1 and CYTB
genes and form distinct monophyletic clades in the phylogenetic tree that
diverged a long time ago (1.049 mya), shortly after
the divergence of Red Slender Loris from the Grey Slender Loris (1.087 mya). The observed sequence variation and divergence time
between the Mysore and the Malabar Slender Lorises are surprisingly high, which
is not very common between primate subspecies. Since they have been evolving
independently for a long period comparable to the divergence time of their
closest species (Loris tardigradus), the
Mysore and Malabar Slender Lorises deserve independent recognition. Moreover,
they occupy a geographically different landscape and unique habitat, where the
Malabar Slender Loris occupies the wet zone of the Western Ghats, while the
Mysore Slender Loris occupies the dry habitat of the eastern slope of the
Western Ghats, dry forests of the Deccan plateau and Eastern Ghats (Kumara et al. 2006, 2009; 2013). They are also
morphologically distinct, where the Malabar Slender Loris appears reddish in
color and almost half the body size of the greyish colored Mysore Slender Loris
(Kumara et al. 2006). Considering these significant
differences in the morphological, geographical, and genetic factors, we propose
to recognize the Mysore Slender Loris Loris lydekkerianus ssp. lydekkerianus
and Malabar Slender Loris Loris lydekkerianus ssp. malabaricus
as two distinct species, Loris lydekkerianus
and Loris malabaricus, respectively.
The divergence time estimates are
supported by a number of molecular markers in the whole mitochondrial genome
and is consistent with previous studies on the evolutionary history of this
genus (Finstermeier et al. 2013). Several
environmental, climatic, and geographical factors might have influenced the
divergence of the Mysore and Malabar Slender Lorises about one million years
ago. The mid-Pleistocene transition (1.25–0.7 Mya) was a time of dramatic
climatic change and glaciation that influenced the environments and
biogeography of Earth (Herbert 2023). The glaciation and interglaciation cycles
affected the sea level, precipitation, temperature, vegetation, and habitat
availability. The environmental conditions in India specifically during the
Pleistocene were diverse and dynamic, ranging from deserts, tropical forests to
grasslands (Morley & Morley 2022). The variability of monsoon coupled with
expansion and contraction of forests due to glacial-interglacial cycles could
have influenced availability of resources, fragmentation of habitats, and
changes in forest cover promoting genetic differentiation and divergence of the
Mysore and Malabar Slender Lorises.
Understanding the genetic
structure and variation of species is crucial for the scientific management of
threatened species and their eventual recovery. The findings of this study have
important implications for the conservation and management of the slender
lorises in India. With a clearer understanding of the genetic differences
between the Mysore and Malabar Slender Lorises, it will be possible to more
accurately identify and classify individual animals, which will in turn
facilitate the development of an effective conservation breeding program. Such
a program can be particularly beneficial for species like the slender loris
that are threatened by habitat loss and fragmentation, and whose populations
have been declining in recent years (Kumara et al. 2006, 2016).
The main drawback of this study
is the limited sample size which we duly acknowledge. It is to be noted that
the construction of whole mitochondrial genomes from WGS data for accurate
molecular dating often requires good-quality DNA from animals of known
geographic origin which is very difficult to obtain, especially for the Malabar
Slender Loris. More samples from the Malabar Slender Loris could better resolve
the phylogenetic tree and nuclear markers could also be used to confirm our
findings and validate the species delimitation. Furthermore, sampling the
individuals from the range edges and the overlapping ranges in the southern
ridge of Western Ghats would provide more statistical power to establish the
monophyly and identify any hybridization. It would also be prudent to include
samples of the Mysore subspecies from Sri Lanka in future studies to fully
comprehend the diversity and understand the evolutionary history of the slender
lorises throughout its geographical range. Comprehensive genome sequencing of
all the subspecies of slender loris would also help to understand the genomic
basis of morphological differences and their adaptations to respective niches.
In conclusion, this study
provides the first molecular evidence for the genetic divergence and
distinctiveness of the Mysore and Malabar Slender Lorises. The sequence
analysis, phylogenetic analyses, and molecular dating suggest that the Mysore
and Malabar Slender Lorises are genetically distinct and have been evolving independently
for a significant period. The high level of genetic divergence between them
highlights the importance of preserving their genetic diversity and underscores
the need for more efforts to conserve them in the wild. By considering the
significant differences in the morphological, geographical, and genetic
factors, we recommend to elevate L.l.
ssp. lydekkerianus and L.l.
ssp. malabaricus to the species level. We
propose to recognize them as two distinct species Loris lydekkerianus
and Loris malabaricus as each of them
represents a unique evolutionary lineage and deserves separate recognition and
protection. We advocate for further studies to validate the species
delimitation with larger sample sizes and recommend for separate conservation
measures and management actions to preserve their unique genetic diversity in
the wild and captivity.
Data Availability Statement
The three whole mitochondrial
genome sequences generated in this study have been submitted to the NCBI
database under the accessions OR115511, OR115512, and OR115513.
Table 1. Sequence distance matrix
of the Loris genus based on full-length cytochrome b and cytochrome
oxidase 1 genes.
|
Sample |
Loris tardigradus-1 |
Loris tardigradus-2 |
Loris lydekkerianus
malabaricus |
Loris lydekkerianus
lydekkerianus-1 |
Loris lydekkerianus
lydekkerianus-2 |
Loris lydekkerianus-Ref |
|
Loris tardigradus-1 |
- |
0 |
2.58 |
2.82 |
2.95 |
2.9 |
|
Loris tardigradus-2 |
0 |
- |
2.58 |
2.82 |
2.97 |
2.9 |
|
Loris lydekkerianus
malabaricus |
2.58 |
2.58 |
- |
2.09 |
2.09 |
2.09 |
|
Loris lydekkerianus
lydekkerianus-1 |
2.82 |
2.82 |
2.09 |
- |
0.16 |
0.08 |
|
Loris lydekkerianus
lydekkerianus-2 |
2.97 |
2.97 |
2.09 |
0.16 |
- |
0.16 |
|
Loris lydekkerianus-Ref |
2.9 |
2.9 |
2.09 |
0.08 |
0.16 |
- |
For figures & image - - click here for full PDF
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Supplementary
file 1. Partitioning scheme and nucleotide substitution model selection output
from PartitionFinder2.
Best
partitioning scheme
Scheme lnL : -64454.10696411133
Scheme AICc : 129409.318896
Number of params : 246
Number of
sites : 13594
Number of subsets : 33
Subset | Best
Model | # sites | Partition names
1 | GTR+I+X | 622
| NAD1_pos1, NAD4l_pos3, ATP6_pos1
2 | TRN+I+X | 533
| NAD1_pos2, ATP6_pos2
3 | HKY+X | 223 | ATP6_pos3
4 | HKY+X | 132 | ATP8_pos1, ATP8_pos2
5 | GTR+X | 317 | ATP8_pos3, COX3_pos3
6 | HKY+G+X | 368
| COB_pos1
7 | GTR+I+X | 368
| COB_pos2
8 | TRN+I+X |
368 | COB_pos3
9 | GTR+G+X | 496
| COX1_pos1
10 | HKY+G+X
| 496 | COX1_pos2
11 | HKY+G+X | 496
| COX1_pos3
12 | K80 | 222 | COX2_pos1
13 | TRN+X | 222 | COX2_pos2
14 | TRN+I+X | 424
| NAD3_pos1, NAD4l_pos2, COX2_pos3
15 | JC | 251 | COX3_pos1
16 | HKY+X | 251 | COX3_pos2
17 | HKY+X | 310 | NAD1_pos3
18 | GTR+I+X | 340
| NAD2_pos1
19 | HKY+I+X | 340
| NAD2_pos2
20 | HKY+I+X | 339
| NAD2_pos3
21 | TRN+I+X | 111
| NAD3_pos2
22 | GTR+I+X | 201
| NAD4l_pos1, NAD3_pos3
23 | HKY+X | 457 | NAD4_pos1
24 | HKY+X | 456 | NAD4_pos2
25 | HKY+I+X | 456
| NAD4_pos3
26 | HKY+I+X | 596
| NAD5_pos1
27 | GTR+I+X | 596
| NAD5_pos2
28 | HKY+I+X | 596
| NAD5_pos3
29 | GTR+G+X | 181
| NAD6_pos1
30 | HKY+I+X | 180
| NAD6_pos2
31 | GTR+G+X | 180
| NAD6_pos3
32 | GTR+G+X | 1454
| rrns
33 | GTR+I+X | 1012
| rrnl