Title
Lorenz Hauser
Jennifer Gardner
Ross Furbush
Abstract:
Molecular markers at cytochrome b reveal trade species composition of Hippocampus supporting the
effectiveness of using genetics on this almost morphologically indistinguishable genus. H. trimaculatus
composes 72% of illegal seizures over the span of 4 years under 12 different shipment accessions with
representation by five additional species. The CITES 10 cm minimum regulation is almost entirely
followed, but only 15% of the seized H. trimaculatus individuals reach measured sexual maturity.
Phylogenetic analysis on haplotype lineages of three species narrows down a region in the Java Sea that
seems to be heavily fished. However, presence of other lineages and H. ingens indicates a far more
complex fishery and trade.
Introduction
Conservation can be more effective when resource managers are able to distinguish priority areas or
populations. When the identity of a declining species is known, conservationists can adjust efforts based
off of known distribution or unique natural history specific to the species. Haplotypes can further
distinguish which populations within the geographic distribution are being greatly reduced. Some
species can be difficult to identify (Hajibabaei 2006) and may require an expert taxonomist to key
specimens to species. Molecular markers, however, such as cytochrome b can frequently be more
helpful in identifying specimens successfully.
Seahorses, the genus Hippocampus, are widely variable in measurements and meristics within and
between species making it difficult to distinguish one from the other based on morphology (Foster
2004). If experts and non-experts can increase accuracy in Hippocampus spp. identification, then
conservation efforts can determine which species is most affected by legal and illegal trade. Analyzing
cytochrome b will more accurately secure Hippocampus spp. identification and help reinforce the
morphological traits that are found within each species.
The charismatic natural history and biology of the seahorse has captured the attention of many markets.
This has ultimately caused a great reduction in population size. Hippocampus spp. are one of the only
fish to swim vertically and occupy a worldwide distribution extending in both tropical and temperate
zones and in both near shore and open ocean habitat. They are patchily distributed and have high site
fidelity (Foster 2004), which increases the impact of the removal of one individual (Hawkins 2003).
Hippocampus spp. life span ranges from 1-5 years depending on the species. Their thick bony plates,
spines, and cryptic nature often protect adult Hippocampus spp. from predation, whereas many larvae
and juveniles are eaten while occupying the planktonic column. The reproductive strategy of the
monogamous Hippocampus spp. receives the most attention from humankind. It is instead the male that
rears the offspring in his pouch after impregnation by a female and her eggs. The attention received
from this unique trait has helped bolster conservation support, but is also the reason Hippocampus spp.
populations are declining (Foster 2004).
The unique reproductive history of Hippocampus spp. attracts the attention of aquarists, the crafts
industry, and the medicinal market. The Chinese and Southeast Asian (India, Thailand, and The
Philippines) are heavily involved in this fishery; China harvests over 25 million Hippocampus
spp.(including shrimp trawl by-catch), 95 percent of which are for traditional medicines. Dried
seahorses are most often used in traditional medicine as an aphrodisiac. Alternative drugs can ease the
pressure on this threatened genus: the introduction of Viagra, a sexual stimulus drug, lowered seahorse
sales by 4.5 percent in Hong Kong in 1998. It is also commonly used as an asthma treatment and less
commonly to lower cholesterol and mend broken bones (Hawkins 2003). The combined pressures from
the previously mentioned markets along with other unmentioned harms such as habitat loss and
changing ocean conditions (Heath 2012) , have caused a population reduction that has increased from
15 to 50 percent from 1990-1995, and continues to rise (Hawkins 2003).
Wildlife trade is regulated by the Convention on International Trade in Endangered Species of Wild
Fauna and Flora (CITES) which includes 160 countries who aim to protect over 34,000 listed CITES
species (Hawkins 2003). From 1998 to 2007, 35 million CITES listed species were exported from
Southeast Asia, 16 million of which were Hippocampus spp.. It was found that Thailand is responsible for
the highest percent of Hippocampus spp. export (94%), and Hong Kong, Taiwan, and China combined are
responsible for highest import (95%) (Nijman 2009). Shipment size in Asian ports has increased
significantly with 40 tons of dried seahorse imports and exports in 1995 and 70 tons in 2000 (Hawkins
2003) where 1 kg of dried seahorses makes up 300 individuals (Nijman 2009).
The current CITES regulation prohibits the trade or transport of any Hippocampus species under 10 cm
in height (Foster 2005). The variation between species height at sexual maturity has conservationists
concerned that some species could possibly be extracted and traded that exceed the minimum, but
remain below reproduction height. The identification of seized, dry Hippocampus spp. will help assess
how effective CITES is with their current regulation and will provide new information on where CITES
can improve in conservation efficiency.
Methods and Materials
Specimen Collection
Dried seahorse specimens (Hippocampus spp.) were obtained from the University of Washington Fish
Collection from 2010-2013. These specimens were confiscated at the border of the United States by
United States Fish and Wildlife Services because these specimens were being brought into the country
without proper CITES documentation. The specifics surrounding the origin and seizure of these
specimens are unknown. These specimens are now to be used for scientific and educational purposes
only.
Morphologic Identification
Attempts were made to identify specimens via morphology using A Guide to the Identification of
Seahorses (Lourie et al. 2004) but this was difficult with dried specimens. Characteristics used for
morphologic identification were: height, head length to snout length ratio, number of tail rings, number
of trunk rings, number of cheek and eye spines, and number of trunk and tail rings supporting the dorsal
fin. In most cases morphological identification to one species was difficult so it was narrowed down to
five possible species. Each specimen was identified by one student in the University of Washington
Seattle’s Genetics and Molecular Ecology course between 2010 and 2013. 244 specimens were
identified via morphology over this period.
DNA Extraction
DNA extraction was done using dorsal fin clips of dried specimens. When dorsal fin clips could not be
obtained tail clippings were taken. Each student was responsible for DNA extraction of the specimen
they identified via morphology.
DNA extraction was done using the QIAGEN DNeasy extraction kit and following the standard protocol.
Incubation for tissue digestion was done at 56⁰ C for 70 minutes. A final elution of 100 ul of DNA was
obtained. DNA elutions were stored at 4⁰ C. DNA was extracted from 244 specimens.
PCR and Visualization
A portion of the Cytochrome b gene (≈900bp) was amplified in a 25 μl PCR reaction using the following
reagents: Qiagen 2x PCR Mix final concentration of 0.9X, 0.2 μM forward and reverse primers, deionized
water, and 2 μl of DNA elution. Seahorse specific primers were used (Lourie and Vincent 2004): Forward:
shf 5′ CTACCTGCACCATCAAATATTTC 3′ and Reverse: shr2 5′ CGGAAGGTGAGTCCTCGTTG 3′. The
thermocycler profile for this reaction was: 95°C initial denaturation for 15 min, 30 PCR cycles of
(denaturation at 94°C for 30 sec, annealing at 57°C for 90 sec, extension at 72°C for 90 sec), followed by
a final extension at 72°C for 10 min. Samples were held at 4°C until removed from the thermocycler.
Visualization of PCR products was done using gel electrophoresis with a 1% agarose gel run at 110V for
77 minutes. Fragment length was compared against Lo Ladder to determine if fragments were correct
length. PCR was successful for 204 specimens.
Sequencing
The 204 PCR products of appropriate length and primers were sent to the High Throughput
Sequencing Center of the Department of Genome Sciences at the University of Washington. The two
PCR primers and Sanger sequencing were used to sequence the product in both directions. Sanger
sequencing was performed using an ABI 3730 automated sequencer (Applied Biosystems, Carlsbad, CA).
Data Analysis
Sequence data from fragments in both directions were returned. Forward and reverse sequences from
one specimen were aligned and trimmed to remove low quality reads using Geneious 7.0.3. Consensus
sequences from pairwise alignments were used to create a full alignment for 186 unknown specimens.
18 specimens returned low quality reads and were not able to be used in further analysis. Full alignment
and identification of sequences was done using Mega 5.2 (Tamura et al. 2011). The muscle alignment
algorithm was used and haplotypes were collapsed using GenAlEx Excel Add-on. Phylogenetic analysis of
haplotypes and known Hippocampus spp. sequences obtained from GenBank was done using maximum
likelihood methods with 1000 bootstraps and the Hasegawa-Kishino-Yano model with gamma
distributed invariant sites. The pipefish (Micropterus brachyurus brachyurus) was used as the out-group.
Phylogeographic analysis of specimens was done using the same sequences that Lourie et al. (2005)
attributed to geographic areas.
Minimum spanning networks of haplotypes for specimens with phylogeographic information were made
using Haplotype viewer (citation). Alignments and trees used for minimum spanning networks were
made using Mega 5.2 (Tamura et al. 2011). Trees were constructed using neighbor-joining methods and
p-distance models with 1000 bootstrap replicates.
Results
186 specimens were successfully identified to species using phylogenetic techniques. For 186 sequences
there were 76 haplotypes. Overall haplotype diversity was 0.9087. Haplotypes were identified as being a
species or subpopulation of a species with bootstrap values of at least 95 (Figure 1). The majority of
specimens and haplotypes were identified as belonging to the H. trimaculatus A subpopulation with
bootstrap support of 100 (Figure 1a). The majority of specimens and haplotypes were identified as
belonging to the H. trimaculatus A subpopulation with bootstrap support of 100 (Figure 1f). Other
species and subpopulations present in this sample included H. spinosissimus A and C subpopulations
(Figure 1b), H. comes (Figure 1c), H. kelloggi (Figure 1d), H.ingens (Figure 1 E), and H. kuda C
subpopution (Figure 1 A) (Lourie et al. 2005).
Phylogeographic results show that the most likely region for the majority of these specimens to have
come from is the Java Sea between the islands of Sumatra and Borneo, north of Java, and south of the
Gulf of Thailand (Figure 2?).
Specimens were received by the UW Fish Collection in shipments from USFWS and each shipment was
assigned an accession number. Genetic results showed that the accessions of 2010-004, 2011-004, and
2011-10 contained specimens of more than one species (Table I). 2010-004 contained specimens of H.
kelloggi and H. trimaculatus. 2011-004 was the most specious containing specimens of H. comes, H.
ingens, H. kuda, and H.trimaculatus. 2011-010 contained specimens of H. spinosissimus and H.
trimaculatus.
Of the 186 identified specimens only two were below the 10 cm height restriction. Both were H.
trimaculatus A. An 89 mm specimen and a 97 mm specimen were found in the 2011-004 and 2010-004
shipments respectively.
A minimum spanning network (MSN) of H. kuda sequences revealed that there divergence between the
A and C subpopulations (Figure 3). The MSN of H. spinosissimus sequences showed divergence between
the A, B, and C subpopulations (Figure 4). The MSN of H. trimaculatus A sequences showed haplotype
radiation around one very common haplotype as well as a secondary radiation around the second most
common haplotype (Figure 4a). A MSN for all of H. trimaculatus was not created due to software
constraints. The MSN of H. trimaculatus B showed a radiation around one haplotype with one very
divergent haplotype (Figure 4b).
Discussion
The species composition of this sample is dominated by H. trimaculatus in almost all shipments.
This could indicate that there is a large H. trimaculatus population, targeted fishery selectivity, or
specific natural history characteristics that make this species easily susceptible to being caught. The
somewhat high frequency of H. comes and the relatively low frequency of the four remaining species,
which are not present for all four years, indicates either low wild population size or low trade pressure
for the reasons previously mentioned for H. trimaculatus. Due to the illegal nature of this accession, the
sample of species could also be indicative of which Hippocampus species are frequently traded
improperly.
The fact that multiple shipments contained multiple specimens is an example of the complexity
of this trade. The ranges of H. trimaculatus, H. comes, H. spinosissimus, H. kelloggi, and H. kuda are all
very similar with an overlap in the Java Sea and Gulf of Thailand region. However, in the 2011-004
shipment there were also H. ingens specimens present. H. ingens comes from the Pacific waters on the
coast of the Americas. The presence of multiple species from different parts of the world in one
shipment shows that shipments are not simply caught and immediately exported. This trade complexity
has been revealed in previous genetic analyses of dried seahorses (Sanders et al. 2008). A key difference
this study reveals is that shipment mixing is happening to some extent before shipments even reach the
border let alone the shop they are being sold in.
Phylogeographic analysis (Lourie et al. 2005) helps to narrow down the area where the majority
of these specimens were caught.
Only 2 of the 186 specimens in this study violated the CITES 10 cm minimum height at export
rule. H. trimaculatus however is one of the few species that reaches reproductive maturity beyond the
CITES regulation height (Table 1). The height at maturity for H. trimaculatus (14 cm) was only reached by
15% of the identified H. trimaculatus specimens and 94% of all other species. This brings into question
the effectiveness of the CITES minimum and whether it properly accounts for the larger Hippocampus
spp. which could, and appear to be, regularly caught before reaching maturity. There seems to be higher
fishing pressure on the larger Hippocampus species as all six species seized and used in this study are
among the 12 largest of all 32 species of Hippocampus.
There is limited information linked to accession number that sheds little light on the
complexities of the trade. Regardless, this sample provides an important data point on the geographic
distribution and species composition of seized individuals.
Acknowledgments
Isadora
Kerry
Other lab TA
Sarah Foster
Qiang (John) Lin
Student of fish 324 from 2010-2013
USFWS
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To Do:
1. Find email with USFWS to properly acknowledge them
2. Haplotype diversity overall