Journal of Threatened
Taxa | www.threatenedtaxa.org | 26 April 2024 | 16(4): 25040–25048
ISSN 0974-7907
(Online) | ISSN 0974-7893 (Print)
https://doi.org/10.11609/jott.8694.16.4.25040-25048
#8694 | Received 14 August 2023 | Final received 27 January 2024 |
Finally accepted 09 April 2024
DNA barcoding reveals a new
population of the threatened Atlantic Forest frog Sphaenorhynchus
canga
Diego J. Santana 1 , André
Yves 2, Elvis A. Pereira 3, Priscila S. Carvalho 4,
Lucio M.C. Lima 5, Henrique C. Costa 6 & Donald B. Shepard 7
1 Instituto de Biociências,
Universidade Federal de Mato Grosso do Sul, Av. Costa
e Silva, s/n, 79070-900, Cidade Universitária,
Campo Grande, Mato Grosso do Sul, Brazil.
2 Programa de Pós-Graduação
em Ecologia, Instituto
Nacional de Pesquisas da Amazônia, Avenida André
Araújo 2936, 69060-001, Petrópolis, Manaus, Amazonas,
Brazil.
3 Departamento de Biologia
Animal, Instituto de Biologia, Universidade
Estadual de Campinas (UNICAMP), Campinas, Brazil.
4 Instituto de Biociências,
Universidade Federal de Mato Grosso do Sul, Av. Costa
e Silva, s/n, 79070-900, Cidade Universitária,
Campo Grande, Mato Grosso do Sul, Brazil.
5 Programa de Pós-Graduação
em Biodiversidade e Conservação da Natureza, Universidade Federal de Juiz de Fora, Rua
José Lourenço Kelmer S/n,
36036-330, Campus Universitário, Juiz de Fora, Minas
Gerais, Brazil.
6 Departamento de Zoologia,
Universidade Federal de Juiz de Fora, Rua José Lourenço Kelmer S/n, 36036-330, Campus Universitário,
Juiz de Fora, Minas Gerais,Brazil.
7 Department of Biological
Sciences, University of Arkansas, 601 Science Engineering Hall, Fayetteville,
Arkansas, 72701, USA.
1 jose.santana@ufms.br
(corresponding author), 2 andreyves7@gmail.com, 3 elvisaps21@gmail.com,
4 pricarvalho.bio@gmail.com, 5 luciobiolima@yahoo.com.br,
6 ccostah@gmail.com, 7 dshep@uark.edu
Abstract: Species identification plays a
significant role in biodiversity conservation. As many species remain
unrecognized, particularly in neotropical hotspots like the Brazilian Atlantic
Forest (AF), novel molecular techniques are being widely employed to bridge
this gap. In this study, we used DNA barcoding and phylogenetic tools to
identify a new population of Sphaenorynchus
canga in the central region of the Brazilian AF.
Our results extend the species’ known distribution by approximately 200 km to
the south, encompassing a different mountain range than its type locality
(Serra do Espinhaço). This disjunct distribution,
while not uncommon among amphibians, suggests a historical connection between
these two mountain complexes as a biogeographic explanation. Despite the
discovery of a new S. canga population, the
species continues to face numerous anthropogenic threats such as mining, land
use, and cattle ranching. Urgent conservation and research efforts are
warranted to ensure the survival of S. canga
populations across these habitats.
Keywords: 16S mtDNA,
Hylidae, Mantiqueira
mountain range, Minas Gerais, species identification.
Abbreviations: ASAP – Assemble Species by
Automatic Partitioning | CAUFJF – Coleção de Anfíbios da Universidade Federal
de Juiz de Fora | ICMBIO – Instituto Chico Mendes de Conservação
da Biodiversidade | IUCN ---– International Union for
Conservation of Nature | MCMC – Markov chain Monte Carlo | ML – Maximum
Likelihood | mtDNA – mitochondrial DNA | PCR –
Polymerase chain reaction | SISBio – Sistema de Autorização e Informação em Biodiversidade | ZUFMS-AMP – Coleção Zoológica da Universidade Federal de Mato Grosso do Sul.
Editor: S.R. Ganesh, Kalinga Foundation, Shivamogga,
India. Date of publication: 26 April 2024
(online & print)
Citation: Santana, D.J., A. Yves, E.A. Pereira, P.S. Carvalho, L.M.C. Lima, H.C.
Costa & D.B. Shepard (2024). DNA barcoding reveals a new population of the
threatened Atlantic Forest frog Sphaenorhynchus
canga. Journal of Threatened Taxa 16(4): 25040–25048. https://doi.org/10.11609/jott.8694.16.4.25040-25048
Copyright: © Santana et al. 2024. 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: Fieldwork was partially funded by Fundação de Amparo à Pesquisa do Estado de Minas Gerais (FAPEMIG, project APQ-02302-21).
Competing interests: The authors declare no competing interests.
Author details: DJS is a professor in the
Zoology Laboratory at the Federal University of Mato Grosso do Sul (UFMS). He is a zoologist with
emphasis in herpetology, working with frogs and reptiles, focusing on topics
such as natural history, phylogenetics, phylogeography,
and biogeography. AY is currently a PhD student at the Universidade
Federal do Paraná. He is an ecologist working on neotropical reptiles and
amphibians, with focus on subjects such as adaptive evolution, landscape
genetics and natural history. EAP is a postdoctoral researcher in the
Laboratory of Natural History of Brazilian Amphibians at the State University
of Campinas (UNICAMP). He is a zoologist with an emphasis on herpetology,
working with amphibians, focusing on topics such as systematics, phylogenetics,
phylogeography, biogeography and conservation. PSC is
a postdoctoral researcher in the Mapinguari Lab at
Federal University of Mato Grosso do Sul (UFMS). She is a zoologist with an
emphasis on reptiles, working with systematics, phylogenetics, phylogeography, biogeography and conservation. LMCL is a
zoologist who has worked on natural history and distribution of reptiles and
amphibians in Brazil. Currently, he directs a private protection area,
supporting initiatives of research, conservation and sustainable tourism. HCC
is a professor at Universidade Federal de Juiz de
Fora, Brazil, researching biogeography, natural history, and taxonomy of
amphibians and reptiles. DBS is a Teaching Associate Professor in the
Department of Biological Sciences at the University of Arkansas, Fayetteville,
Arkansas, USA. His research employs molecular methods and geospatial tools to
examine patterns of genetic variation, identify cryptic diversity, and
understand the processes that drive diversification of amphibians and reptiles.
Author contributions: Field work: AY, DJS, EAP, HCC,
LMCL, PSC; data analysis: DJS, SBS, PSC; writing: all authors; revisions: all
authors.
Acknowledgments: We thank Fundação
de Amparo à Pesquisa do Estado de Minas Gerais
(FAPEMIG process APQ-02302-21) and the Institutional Program of
Internationalization sponsored by Coordination for the Improvement of Higher
Education Personnel (Capes-PrInt 41/2017 – Process
88881.311897/2018–01) for financial support. DJS thanks CNPq
for his research fellowships (CNPq 309420/2020-2; CNPq 402012/2022-4). Lab work and DNA sequencing were
supported by Dr. Johnny Armstrong and the Louisiana Tech University School of
Biological Sciences. Collections were authorized by the System of Authorization
and Information in Biodiversity, Ministry of the Environment (license SISBIO
45889).
Species identification is a
crucial component of biodiversity research and conservation (Delić et al. 2017; Lyra et al. 2017; Sheth
& Thaker 2017). To this end, DNA barcoding has
become a widely used molecular technique for identifying species. This approach
relies on sequencing a standardized fragment of DNA that can be compared to
reference databases to accurately identify species (Gehara
et al. 2013; Koroiva & Santana 2022). DNA
barcoding has also proven to be effective in delimiting species, and it has
been applied across a wide range of taxa, including amphibians (Jansen et al.
2011; Koroiva et al. 2020; Koroiva
& Santana 2022).
Delimitation and identification
of amphibians using robust methods is paramount, given that they are the most
threatened group of terrestrial vertebrates worldwide (Howard & Bickford
2014; Cox et al. 2022; Toledo et al. 2023). Many species of amphibians are
being classified under the IUCN Red List categories at the same time they are
being formally named (Brasileiro et al. 2007; Caramaschi & Cruz 2011; Assis et al. 2013). Atlantic
Forest, a biodiversity hotspot (Myers et al. 2000; Ribeiro et al. 2011; Zachos & Habel 2011), harbors
more than 625 amphibian species, 77% of them endemic, and many with very narrow
distributions (Rossa-Feres et al. 2017). Since the
arrival of the first European colonizers in the early 16th century,
the Atlantic Forest has lost most of its original cover, and the remaining is
heavily fragmented (Ribeiro et al. 2009, 2011). In Brazil, habitat loss is the
main threat to amphibians living in this rainforest (ICMBio
2018); 41 species are in peril, and two are already declared extinct (Ministério do Meio Ambiente 2022). The Atlantic Forest is also the region with
the highest amphibian population declines reported worldwide (Toledo et al.
2023). Therefore, identifying and describing the amphibian diversity of the
Atlantic Forest is crucial for its conservation and for developing
targeted conservation strategies.
The landscape of the southeastern
portion of the Atlantic Forest includes many mountain ranges that are
considered cradles of amphibian diversity (Leite et
al. 2008; Neves et al. 2018; Silva et al. 2018). These mountain chains harbor
most of the endemic amphibian species from the Atlantic Forest (Guedes et al.
2020), and many are threatened (Pontes & Guidorizzi
2023). One such species is the Hatchet-faced Canga
Lime Treefrog, Sphaenorhynchus canga, first described in 2015 (Araujo-Vieira et al.
2015) and known
only from a small area in the southern portion of the Espinhaço
Mountain range in Minas Gerais (Silveira et al. 2020). The species is classified
by the Brazilian Ministry of Environment as Critically Endangered (Ministério do Meio Ambiente 2022; Pontes & Guidorizzi
2023).
During field expeditions in the
northern portion of the Mantiqueira Mountain range in
southern Minas Gerais in December 2015, January 2020, and November 2021, a
series of specimens of Sphaenorhynchus were
collected. We collected five adult male specimens during visual and acoustic
searches in one pond in the countryside of Bom Jardim
de Minas, Minas Gerais (-22.004, -44.180; 1,210 m; datum = SAD69). Specimens were euthanized in a 2% lidocaine chlorhydrate solution (MCTIC 2018), fixed in 10% formalin,
and preserved in 70% alcohol. Prior to fixation, we collected tissue samples
(muscle and liver) and stored them in cryotubes filled with 100% ethanol.
Voucher specimens and tissues were deposited in the Coleção
de Anfíbios da Universidade
Federal de Juiz de Fora (CAUFJF), Juiz de Fora municipality, Minas Gerais, and
in the Coleção Zoológica da
Universidade Federal de Mato Grosso do Sul
(ZUFMS-AMP), Campo Grande municipality, Mato Grosso do Sul, Brazil. Collection
permits for this study were issued by ICMBIO (SISBio
73975-1 and 72874-1).
DNA was extracted using the QIAGEN DNeasy Blood and Tissue Kit (Valencia, California, USA)
following the manufacturer’s protocol. Next, a fragment of the mitochondrial
16S gene was amplified using primers 16Sar and 16Sbr (Palumbi
et al. 2002). The PCR protocol was configured with one initial phase of 94°C
for 3 min, followed by 35 cycles of 94°C for 20s, 50°C for 20s, 72°C for 60s,
and a final extension phase of 72°C for 5 min. Purification of PCR products and
sequencing were performed by Eurofins Genomics Inc. (Louisville, Kentucky,
USA). Comparable 16S sequences of Sphaenorhynchus
from GenBank and one sequence of Scinax fuscovarius to use as an outgroup were downloaded (Supplementary Table 1).
All 16S mtDNA gene fragments were aligned using the
MAFFT algorithm (Katoh & Toh
2008) in Geneious v9.0.5 with default settings. The
final dataset comprised 53 sequences of a 515 base-pair (bp)
fragment of the 16S gene. A maximum likelihood tree was inferred in RAxML (Stamatakis 2014) via raxmlGUI 2.0 (Edler et al. 2021).
The analysis was conducted using a ML + rapid bootstrap setting with a GTR+I+G
substitution model and 1,000 bootstrap replicates. The appropriate substitution
model was confirmed with Modeltest (Darriba et al. 2020) in raxmlGUI
2.0. Additionally, PTP and bPTP species delimitation
analyses were conducted (Zhang et al. 2013) using the ML Tree. Calculations
were performed on PTP webserver (http: //species.h-its.org/ptp/)
with 500,000 MCMC generations, thinning set at 100, and burn-in at 10%. In
addition, we performed the delimitation method Assemble Species by Automatic
Partitioning (ASAP) on the online server
(https://bioinfo.mnhn.fr/abi/public/asap/asapweb.html) using a simple distance
model to compute distances between samples and default parameters (Puillandre et al. 2021). To explore relationships among mtDNA haplotypes, we estimated a 16S haplotype network
among species closely related to S. canga—S.
botocudo, S. cammaeus,
S. caramaschii, S. platycephalus,
and S. surdus (Pereira et al. 2022)—in POPART
(Leigh & Bryant 2015) using the median-joining network method. We depict
each species using different colors in the haplotype network. Lastly, we
calculated sequence divergence (uncorrected p-distance) among
species/individuals using MEGA v10.1.1 (Kumar et al. 2018).
We identified the Sphaenorhynchus from Bom
Jardim de Minas as Sphaenorhynchus canga (Image 1). Our maximum likelihood tree (Figure 1)
of the mitochondrial 16S gene confidently (bootstrap = 0.98) placed the
sequenced specimens with Sphaenorhynchus canga, sister to a clade formed by S. botocudo and S. surdus.
The three species delimitation methods we used yielded the same results,
recovering one evolutionary entity for each known species (Figure 1). All three
analyses confidently recovered all populations of Sphaenorhynchus
canga as a single evolutionary lineage. Our
haplotype network (Figure 2) shows a clear separation between all species of Sphaenorhynchus. The genetic distance between S. canga from Bom Jardim de
Minas and S. canga from the type locality was
0.4% (Supplementary Table 2). Overall, the morphology of S. canga from Bom Jardim de
Minas also have the standard diagnosis of the species presented in its original
description, such as the lack of tympanic membrane, the snout protruding in
profile, the presence of a canthal white line, a dorsolateral white line from
the eye to sacral region, and a dorsolateral black line from the tip of snout
extending beyond the eye and disappearing up to the flanks (Araujo-Vieira et
al. 2015). The newly discovered population of S. canga
in Bom Jardim de Minas extends the distribution of
the species by about 200 km southward to a different mountain range, Serra da Mantiqueira (Image 2).
The distribution of S. canga in both the southern Espinhaço
and the northern Mantiqueira mountain ranges is a
pattern observed in other anuran species as well, including Bokermannohyla
feioi, Pithecopus
ayeaye, Physalaemus
maximus, and Scinax tripui
(Baêta et al. 2007; Magalhães
et al. 2017; Silveira et al. 2019; Brunes et al.
2023). This shared distribution pattern has led biogeographers to hypothesize a
historical connection between the Espinhaço and Mantiqueira mountain ranges (Magalhães
et al. 2017; Neves et al. 2018; Brunes et al. 2023).
The discovery of S. canga in the Mantiqueira Mountains adds additional support for a
historical connection between these mountain ranges and increases the potential
area where this species could occur, particularly in protected areas in the
northern portion of Serra da Mantiqueira.
While the International Union for
Conservation of Nature (IUCN) Red List of Threatened Species classifies this
species as of Least Concern (IUCN & Boitatá
2023), the latest update of the Brazilian Red List, which is based on IUCN
criteria, classifies Sphaenorhynchus canga as Critically Endangered (Pontes & Guidorizzi 2023) due to its limited geographic distribution
and restriction to ponds in ironstone outcrops, a habitat severely impacted by
mining (Bastos et al. 2022). The IUCN assessment states that ‘there are no
ongoing major threats, the species is a habitat generalist occurring even in
modified areas, and it is presumed to have a large and stable population’ (IUCN
& Boitatá 2023). However, mining activity poses a
significant threat to S. canga. Five out of
six ponds where the type series was collected are influenced by mining
activities (Pena et al. 2017). Mining activity has resulted in a continuous
decline in both the area and quality of S. canga’s habitat due to the suppression
of ironstone outcrops and vegetation (Bastos et al. 2022). Recent surveys have
had some success in finding the species in additional localities and habitats,
including perennial small dams and anthropogenic swamps inside or on the edge
of semi-deciduous seasonal forests, suggesting that it may have some degree of
ecological plasticity; nonetheless, the species’ spatial extent has only
slightly increased because of these discoveries (Silveira et al. 2020). The
discovery of S. canga in Bom
Jardim de Minas is an important contribution to the conservation of this
species, as research on its geographic distribution is among the main
priorities (Bastos et al. 2022). Although our discovery has increased the
species’ distribution by more than 200 km (straight-line distance), it is important
to note that is still restricted to high-elevation areas, reproducing in ponds
(Araujo-Vieira et al. 2015; Silveira et al. 2020). Furthermore, no known
populations of the species occur in protected areas (Bastos et al. 2022; this
study).
While this discovery provides a
glimmer of hope for the species, it is essential to note that the new
population was found adjacent to a dirt road and in areas designated for cattle
ranching, where vegetation around marshes is typically burned annually by local
farmers. Moreover, the region is experiencing an increase in real estate
speculation for allotments, and a proposal is currently under consideration for
the installation of a hydroelectric power plant at the Pacau
waterfall (Cachoeira do Pacau),
just 5 km from the discovered population. Therefore, future visits to the
locality are of utmost importance to monitor this population and to search for
additional areas where the species may be present. A reevaluation of the
conservation status of S. canga based on this
discovery is beyond the scope of this work. Nonetheless, it is evident that
this finding underscores the urgent need for further research, conservation
measures, and advocacy efforts to ensure the survival of this critically
endangered species.
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Supplementary Table 1.
GenBank accession numbers for mitochondrial 16S gene sequences of specimens of Sphaenorhynchini (Sphaenorhynchus
and Gabohyla) and the outgroup Scinax fuscovarius
included in the molecular analyses.
|
Species |
Genbank accession number |
Reference |
|
S. botocudo |
KY418014 |
Roberto et al. (2017) |
|
S. botocudo |
MK266722 |
Araujo-Vieira et al. (2019) |
|
S. botocudo |
MK266723 |
Araujo-Vieira et al. (2019) |
|
S. botocudo |
MK266724 |
Araujo-Vieira et al. (2019) |
|
S. botocudo |
MK266725 |
Araujo-Vieira et al. (2019) |
|
S. cammaeus |
KY418013 |
Roberto et al. (2017) |
|
S. cammaeus |
MK266726 |
Araujo-Vieira et al. (2019) |
|
S. cammaeus |
MK266727 |
Araujo-Vieira et al. (2019) |
|
S. canga |
KY418015 |
Roberto et al. (2017) |
|
S. canga |
HCC193 |
Present Work |
|
S. canga |
HCC194 |
Present Work |
|
S. canga |
MAP6807 |
Present Work |
|
S. canga |
MK266728 |
Araujo-Vieira et al. (2019) |
|
S. caramaschii |
KP096219 |
Araujo-Vieira et al. (2015) |
|
S. caramaschii |
KP096220 |
Araujo-Vieira et al. (2015) |
|
S. caramaschii |
MK266729 |
Araujo-Vieira et al. (2019) |
|
S. caramaschii |
MK266730 |
Araujo-Vieira et al. (2019) |
|
S. caramaschii |
MK266731 |
Araujo-Vieira et al. (2019) |
|
S. caramaschii |
MK266732 |
Araujo-Vieira et al. (2019) |
|
S. caramaschii |
MK266733 |
Araujo-Vieira et al. (2019) |
|
S. caramaschii |
MK266734 |
Araujo-Vieira et al. (2019) |
|
S. caramaschii |
MK266735 |
Araujo-Vieira et al. (2019) |
|
S. caramaschii |
MK266736 |
Araujo-Vieira et al. (2019) |
|
S. caramaschii |
MK266737 |
Araujo-Vieira et al. (2019) |
|
S. caramaschii |
MK266738 |
Araujo-Vieira et al. (2019) |
|
S. caramaschii |
MK266739 |
Araujo-Vieira et al. (2019) |
|
S. carneus |
MK266740 |
Araujo-Vieira et al. (2019) |
|
S. carneus |
MK266741 |
Araujo-Vieira et al. (2019) |
|
S. dorisae |
AY843766 |
Faivovich et al. (2005) |
|
S. dorisae |
MK266742 |
Araujo-Vieira et al. (2019) |
|
S. lacteus |
AY549367 |
Faivovich et al. (2004) |
|
S. lacteus |
JF790143 |
Jansen et al. (2011) |
|
S. lacteus |
JF790144 |
Jansen et al. (2011) |
|
S. lacteus |
MK266743 |
Araujo-Vieira et al. (2019) |
|
S. lacteus |
MK266744 |
Araujo-Vieira et al. (2019) |
|
S. mirim |
MK266745 |
Araujo-Vieira et al. (2019) |
|
G. pauloalvini |
MK266747 |
Araujo-Vieira et al. (2019) |
|
G. pauloalvini |
MK266748 |
Araujo-Vieira et al. (2019) |
|
G. pauloalvini |
MK266749 |
Araujo-Vieira et al. (2019) |
|
G. pauloalvini |
MK266750 |
Araujo-Vieira et al. (2019) |
|
G. pauloalvini |
MT503969 |
Orrico et al. (2021) |
|
S. planicola |
MK266751 |
Araujo-Vieira et al. (2019) |
|
S. platycephalus |
KY418016 |
Roberto et al. (2017) |
|
S. platycephalus |
MK266746 |
Araujo-Vieira et al. (2019) |
|
S. prasinus |
MK266752 |
Araujo-Vieira et al. (2019) |
|
S. prasinus |
MK266753 |
Araujo-Vieira et al. (2019) |
|
S. prasinus |
MK266754 |
Araujo-Vieira et al. (2019) |
|
S. surdus |
KY418017 |
Roberto et al. (2017) |
|
S. surdus |
MK266755 |
Araujo-Vieira et al. (2019) |
|
S. surdus |
MK266756 |
Araujo-Vieira et al. (2019) |
|
S. surdus |
MK266757 |
Araujo-Vieira et al. (2019) |
|
S. surdus |
MK266758 |
Araujo-Vieira et al. (2019) |
|
Scinax fuscovarius |
MK266760 |
Araujo-Vieira et al. (2019) |
Supplementary Table 2.
Average uncorrected (p-distance) sequence divergence between different
species of Sphaenorhynchus. Values in bold
along the diagonal are intraspecific divergences. n/c= not calculated.
|
|
|
1 |
2 |
3 |
4 |
5 |
6 |
7 |
8 |
9 |
10 |
11 |
12 |
13 |
|
1 |
S. botocudo |
0.004 |
|
|
|
|
|
|
|
|
|
|
|
|
|
2 |
S. cammaeus |
0.067 |
0.000 |
|
|
|
|
|
|
|
|
|
|
|
|
3 |
S. canga |
0.039 |
0.055 |
0.004 |
|
|
|
|
|
|
|
|
|
|
|
4 |
S. caramaschi |
0.073 |
0.073 |
0.059 |
0.006 |
|
|
|
|
|
|
|
|
|
|
5 |
S. carneus |
0.151 |
0.140 |
0.143 |
0.137 |
0.004 |
|
|
|
|
|
|
|
|
|
6 |
S. dorisae |
0.133 |
0.140 |
0.121 |
0.137 |
0.167 |
0.002 |
|
|
|
|
|
|
|
|
7 |
S. lacteus |
0.107 |
0.102 |
0.106 |
0.099 |
0.137 |
0.090 |
0.005 |
|
|
|
|
|
|
|
8 |
S. mirim |
0.166 |
0.157 |
0.149 |
0.156 |
0.183 |
0.126 |
0.127 |
n/c |
|
|
|
|
|
|
9 |
S. platycephalus |
0.053 |
0.054 |
0.032 |
0.060 |
0.140 |
0.132 |
0.102 |
0.135 |
0.000 |
|
|
|
|
|
10 |
S. pauloalvini |
0.114 |
0.092 |
0.107 |
0.097 |
0.130 |
0.128 |
0.107 |
0.149 |
0.096 |
0.006 |
|
|
|
|
11 |
S. planicola |
0.147 |
0.139 |
0.127 |
0.135 |
0.162 |
0.131 |
0.122 |
0.065 |
0.112 |
0.132 |
n/c |
|
|
|
12 |
S. prasinus |
0.110 |
0.089 |
0.087 |
0.090 |
0.137 |
0.125 |
0.091 |
0.142 |
0.090 |
0.096 |
0.144 |
0.007 |
|
|
13 |
S. surdus |
0.032 |
0.045 |
0.023 |
0.060 |
0.140 |
0.135 |
0.107 |
0.157 |
0.028 |
0.105 |
0.139 |
0.092 |
0.000 |