Hidden introductions of freshwater red algae via the aquarium trade exposed by DNA barcodes

Abstract

The global aquarium trade can introduce non-native invasive freshwater organisms, which can impact local aquatic ecosystems and their biodiversity. It is unassessed whether the aquarium trade spreads freshwater red macroalgae that hitchhike on ornamental aquatic plants and animals. We investigated this via a broad biodiversity survey and genetic analysis of freshwater red algae in the field and aquarium shops in East Asia. Using rbcL-based DNA barcoding, we surveyed 125 samples from 46 field sites and 88 samples from 53 aquarium shops (213 samples in total) mostly across Taiwan—a key hub in the global aquarium trade—as well as in Hong Kong, Okinawa (Japan), the Philippines, and Thailand. We augmented our rbcL sequences with GenBank rbcL sequences that represent 40 additional countries globally. We found 26 molecular operational taxonomic units (mOTUs), some of which are cryptic, in Taiwan. Phylogeographical analysis revealed three potential introduced mOTUs,
which exhibit no local genetic variation in Taiwan and are distributed across continents. Also, we posit that aquaria may serve as an unintentional ex situ conservation site for freshwater red algae that are vulnerable to water pollution due to anthropogenic disturbances. Collectively, these data suggest that freshwater red algae have been hitchhiking and dispersed via the aquarium trade, an important and overlooked mechanism of introduction of the organisms across the globe.

Full text

Environmental DNA. 2021;3:481–491.
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481wileyonlinelibrary.com/journal/edn3
1 | INTRODUCTION
The aquarium trade facilit ates introduction of non-native spe-
cies and homogenization of biodiversity in aquatic ecosystems
worldwide (e.g., Padilla & Williams, 200 4; Rahel, 2007; Strechker
et al., 2011). Many introduced species have hardly discernible
or subtle impacts on the biodiversity of local environments, but
some might become harmful invasive species (Strayer, 2010;
Simberloff, 2014). Efforts to eradicate invasive species incur sub-
stantial social and economic costs, yet they are often unsuccess-
ful (reviewed in Simberloff et al., 2013). Therefore, to prevent and
control the spread of invasive species, it is important to establish
an early warning and rapid response system. To support the im-
plementation of such a system, knowledge about the t axonomic
diversit y and transportation of potential invasive species is criti-
cally needed.
Received: 14 July 2020
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Revised: 7 Se ptember 2020
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Accepted: 7 Septembe r 2020
DOI: 10.10 02/ed n3.139
ORIGINAL ARTICLE
Hidden introductions of freshwater red algae via the aquarium
trade exposed by DNA barcodes
Shing Hei Zhan1 | Tsai-Yin Hsieh2 | Lan-Wei Yeh2 | Ting-Chun Kuo3 |
Shoichiro Suda4 | Shao-Lun Liu2
This is an op en access article under t he terms of the Creat ive Commons Attributio n License, which permits use, dist ribution and reproduc tion in any medium,
provide d the orig inal work is proper ly cited .
© 2020 The Authors . Environmental DNA published by Joh n Wiley & Sons Ltd
1Depar tment of Zo ology a nd Biodi versit y
Research Centre , University of Br itish
Columbia, Vancouver, BC, Canada
2Depar tment of L ife Science and Center
for Ecolog y and Environment, Tunghai
University, Taichung, Taiwan
3Instit ute of Marine Affa irs and Re sources
Management, National Taiwan Ocean
University, Keelung City, Taiwan
4Department of Chemistry, Biology
and Mari ne Science, Facult y of Scien ce,
University of the Ryuk yus, Nishihara , Japan
Correspondence
Shao-Lun Liu, De partment of Life Science
and Cente r for Ecology and Environment,
Tunghai University, Taichung 40704, Taiwan.
Email:shaolunliu@thu.edu.tw; shaolunliu@
gmail.com
Funding information
Ministry of Science and Technology, Taiwan,
Grant /Award Numbe r: MOST108 -2621-B-
029-005-MY3
Abstract
The global aquarium trade can introduce non-native invasive freshwater organisms,
which can impact local aquatic ecosystems and their biodiversity. It is unassessed
whether the aquarium trade spreads freshwater red macroalgae that hitchhike on
ornamental aquatic plants and animals. We investigated this via a broad biodiversity
survey and genetic analysis of freshwater red algae in the field and aquarium shops
in East Asia. Using rbcL-based DNA barcoding, we surveyed 125 samples from 46
field sites and 88 samples from 53 aquarium shops (213 samples in total) mostly
across Taiwan—a key hub in the global aquarium trade—as well as in Hong Kong,
Okinawa (Japan), the Philippines, and Thailand. We augmented our rbcL sequences
with GenBank rbcL sequences that represent 40 additional countries globally. We
found 26 molecular operational taxonomic units (mOTUs), some of which are cryptic,
in Taiwan. Phylogeographical analysis revealed three potential introduced mOTUs,
which exhibit no local genetic variation in Taiwan and are distributed across conti-
nents. Also, we posit that aquaria may serve as an unintentional ex situ conservation
site for freshwater red algae that are vulnerable to water pollution due to anthropo-
genic disturbances. Collectively, these data suggest that freshwater red algae have
been hitchhiking and dispersed via the aquarium trade, an important and overlooked
mechanism of introduction of the organisms across the globe.
KEYWORDS
aquarium trade, DNA barcodes, freshwater red algae, introduction, invasion

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As invasive organisms, aquatic hitchhikers are thought to be
damaging to aquatic ecosystems (e.g., Patoka et al., 2016; Duggan
& Pullan, 2017; Duggan et al., 2018). The diversity and introduction
potential of aquatic hitchhikers in the aquarium trade have been
overlooked, mainly because of their morphological crypticity (e.g.,
invertebrates hitchhiking on ornamental plants;Patoka et al., 2016;
Duggan et al., 2018). Moreover, while the term “hitchhikers” can
refer to organisms that are transported by inanimate vehicles such
as ships and airplanes (Hulme, 2009), in this study we use the term
to refer to organisms that are transported while being attached to
animals or plants.
Some freshwater red macroalgae are aquatic hitchhikers that
are occasionally found in aquarium tanks (e.g., Kaufmann, 2010),
but they are rare in the field near cities due to their vulnerability
to polluted water (Sheath & Hambrook, 1990; Sheath & Vis, 2015).
These algae produce inconspicuous spores that suspend in water or
adhere to aquatic organisms, such as aquatic plants (Figure 1a), cray-
fish (Figure 1b), and snails (Figure 1c-e). Spores can be generated
by asexual reproduc tion (when the algae are turf-like sporophytes,
or chantransia) or by sexual reproduction (when the algae appear
as thread-like, mucilaginous gametophytes). The asexual form may
facilitate the population establishment of introduced freshwater red
algae in the non-native range where mating partners may be unavail-
able or scarce, but to our knowledge this has not been supported
empirically.
Only a few freshwater algal species have been documented as
biological invasions. These known invaders are Bangia atropurpurea
(a red alga; Lin & Blum, 1977), Chara connivens (a green alga;
Luther, 1979), Nitellopsis obtusa (a gr een alg a; Sch loe ss er et al. , 198 6),
Compsopogon caeruleus (a red alga; Manny et al., 1991), Hydrodic tyon
reticulatum (a green alga; Hawes et al., 1991), and Ulva flexuosa (a
green alga; Kaštovský et al., 2010). These algae have been intro-
duced by discharge of ballast water (e.g., Lin & Blum, 1977; Manny
et al., 1991) or accidental release from laborator y experiments
(Hawes et al., 1991). Freshwater red macroalgae can be dispersed
through the aquarium trade (Composopogon caeruleus, Stoyneva
et al., 2006; Montagnia macrospora, Kato et al., 2009). Recently, it
has been speculated that the cosmopolitan distribution of freshwa-
ter red macroalgae may be facilitated by the global aquarium trade
(e.g.,Carlile & Sher wood, 2013; Johnston et al., 2018).
Traditional biodiversity monitoring programs depend on mor-
phology-based approaches, which require time-consuming di-
agnostics by taxonomy experts (Riedel et al., 2013). Hitchhiking
freshwater red macroalgae are morphologically indistinguishable,
thus rendering their species identification challenging. A power-
ful alternative approach to monitor biodiversity is DNA barcoding,
which typically involves sequencing individual genetic loci. DNA
barcodes have been applied to detect introduced and invasive or-
ganisms (Armstrong & Ball, 2005; Pečnikar & Buzan, 2014). For ex-
ample, Vranken et al. (2018) investigated the spread of non-native
marine macroalgae (i.e., seaweeds) via the aquarium trade in Europe
using DNA barcodes. To our knowledge, no study has examined the
introduction of freshwater red macroalgae via the aquarium trade at
a geographically broad scale.
FIGURE 1 Examples of epiphytic (a) and epizoic (b-e) freshwater red macroalgae as aquatic hitchhikers (black arrows). (a) Compsopogon
coeruleus (THU.369) (black arrow) on an ornamental aquatic plant, Aubulias barteri (white arrow), in the Gou-Lao-Ban Pet Shop (Taoyuan,
Taiwan). (b) The chantransia stage of Sheathia dispersa (THU.575) (black arrow) on a red swamp crayfish, Procambarus clarkii (white arrow), in
the Yu-Zhong-Yu Aquarium Shop (Taichung, Taiwan). (c) The chantransia stage of Montagnia macrospora (THU.537) (black arrow) on an apple
snail, Pomacea canaliculata (white arrow), in Nanshi River (Taichung, Taiwan). (d) The chantransia stage of Montagnia macrospora (THU.401)
(black arrow) on a chopstick snail, Stenomelania sp. (white arrow), in the Mataian Wetland Ecological Park (Hualien, Taiwan). (e) A six-month
laboratory culture showing the growth of the gametophyte of S. dispersa (black arrow) on the shell of a chopstick snail, Stenomelania sp.
(white arrow), which was collected from the field [Colour figure can be viewed at wileyonlinelibrar y.com]
(a) (b)
(c) (d) (e)

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ZHAN et A l.
Besides species identification, DNA barcode sequence data can
be utilized to estimate the effect of introduction(s) on the genetic
diversit y of a non-native species (e.g., Bonett et al., 2007; Kinziger
et al., 2011). The population of a newly introduced species is pre-
dicted to harbor lower genetic diversity than its source population(s)
as a consequence of a recent genetic bot tleneck (Nei, Maruyama,
& Chakraborty, 1975; Barrett & Husband, 1990), but multiple intro-
ductions may increase the genetic diversity of an introduced popu-
lation (Novak & Mack, 1993, 20 05) (empirical evidence reviewed in
Dlugosch & Parker, 2008). Following this logic, the genetic informa-
tion in DNA barcodes, in combination with geographic occurrence
data, can enable detection of putative introduced or invasive spe-
cies. Indeed, DNA barcode markers, which exhibit population-level
genetic variation in the red algae (such as rbcL), have been applied
to identif y the source of introduction of marine red algal species
of Polysiphonia (e.g., McIvor et al., 2001; Kim et al., 2004; Geoffroy
et al., 2012, 2016).
In this study, we employed DNA barcoding to survey the biodi-
versity of freshwater red macroalgae from the field and aquarium
shops across Taiwan—a key hub in the global aquarium trade (Padilla
& Williams, 2004)—as well as in a few surrounding regions (Hong
Kong, Okinawa in Japan, the Philippines, and Thailand). We collected
213 ma c roa l gal sp eci men s fro m 99 si tes (mos tly in Taiwa n , 196 sp eci -
mens from 90 sites), determined their rbcL sequences, and estimated
the number and species identity of molecular operational taxonomic
units (mOTUs). Although various genetic markers have been used
to track introduced red algae (rbcL, e.g., McIvor et al., 2001, Mineur
et al., 2012, and Hoffman et al., 2015; rbcL spacer, cox2-3 spacer, and
LSU, e.g., Dijoux et al., 2014; cox1 and rbcL, e.g., Montes et al., 2017),
we chose the plastid gene rbcL because the global sampling coverage
of published rbcL sequences is better than the other genetic mark-
ers for freshwater red algae (see Table S1 for the full list of publi-
cations; also available online in Dryad: doi:ht tps://doi.org/10.5061/
dryad.3n5tb 2rf8). These data yielded, for the first time, a detailed
picture of the biodiversity of freshwater red macroalgae in Taiwan—
in the field and in aquaria. To identif y potential introduced species in
Taiwan, we examined the genetic diversity and global distribution of
the mOTUs by analyzing our data jointly with previously published
rbcL sequences of specimens collected worldwide. Also, we tested
whether the asexual or sexual form of freshwater red algae is more
frequently obser ved in the field or aquaria. Finally, we analy zed or-
namental fish trade data to assess the plausibility of an introduction
route of an alga via Taiwan's aquarium trade.
2 | MATERIALS AND METHODS
2.1 | Sample collection
In total, 213 specimens were collected from the field and aquarium
shops in Taiwan, Okinawa (Japan), Hong Kong, the Philippines, and
Thailand (Figure 2; Table S1); of the 213 samples, 125 were col-
lected from 46 field sites (45 stream or spring sites and one estu-
ary site) and 88 from 53 aquarium shops. After collection, a portion
of the material (about 100–200 mg) was preserved in silica gel or
95% ethanol for molecular analyses, whereas the rest of the mate-
rial was preserved in 10%–15% formalin solution for morphological
examinations.
2.2 | DNA extraction, PCR, and Sanger sequencing
DNA was extracted from silica gel-dried specimens or 95% etha-
nol-preserved specimens by using the commercial ZR Plant/Seed
DNA kit (Zymo Research, CA, USA) following the manufacturer's
protocol. We amplified rbcL under the PCR conditions described
in the protocol of Wang et al. (2005) using these gene-specific
primers: two forward (F7: 5'-AACTCTGTAGAACGNACA AG-3'
and F160: 5'-CCTCAACCAGGAGTAGATCC-3') and one reverse
(R753: 5'-GCTCT TTCATACATATCTTCC-3') ( Vis et al., 1998; Lin
et al., 2001). The PCR products were sent to the Mission Biotech
Company (Taipei, Taiwan) for Sanger sequencing. The GenBank ac-
cessions of the newly generated sequences are listed in Table S1.
2.3 | Sequence acquisition and curation
Poor taxon sampling may lower the accuracy of phylogenetic tree re-
construction and species delimitation (Esselstyn et al., 2012; Ahrens
et al., 2016). Therefore, we combined the newly generated rbcL
FIGURE 2 Sites in Taiwan, Okinawa
(Japan), Hong Kong, Thailand, and the
Philippines sampled in this study [Colour
figure can be viewed at wileyonlinelibrary.
com]

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sequences with additional rbcL sequences from NCBI GenBank (ac-
cessed on 18 October 2018). Using the literature as a guide (see the
references in Table S1), we downloaded from GenBank 1,046 rbcL
sequences of the freshwater and marine species of the following
genera: Bangia, Bostrychia, Caloglossa, and Hildenbrandia. We built a
preliminary phylogeny to identify potential contaminant sequences,
which appear as either unexpected nonmonophyletic placements
or long branch attraction. The following 12 potential contaminant
GenBank sequences were removed: ten Hildenbrandia sequences
(AF107812, AF107818, AF107822, AF107827 to AF107831,
AF534406, AF534408), one Thorea sequence (GU953248), and one
Bangia sequence (AF043379).
The 1,034 cleaned GenBank sequences and the 213 newly
produced sequences (deposited in GenBank under the acces-
sions: MH835465-MH835677) were combined into a dataset of
1,247 sequences (1,054 freshwater taxa and 193 nonfreshwater
taxa). We reduced sequence redundancy by removing identical
sequences using the UCLUST function (hereafter, UCLUST100)
implemented in USEARCH v8.1.1756 (Edgar, 2010). From each
UCLUST100 cluster, we took the longest sequence; in cases of a
tie, one of the longest sequences was picked at random. This re-
sulted in a final dataset of 639 sequences (500 freshwater taxa,
135 nonfreshwater taxa, and 4 taxa that can occur in either fresh-
water or nonfreshwater habitats) for the phylogenetic analysis
and species delimitation. The sequences were mostly contrib-
uted by certain geographical regions—including Australia, Brazil,
the United States, Europe, Japan, and Taiwan (primarily from this
study)—where there has been extensive taxonomic diversity work
(Figure S1). Therefore, the biodiversity reported in this study is
biased heavily toward those regions (Figure S2).
We updated species names by consulting the taxonomic nomen-
clature in AlgaeBase (Guiry & Guiry, 2020; accessed 7 March 2020).
2.4 | Phylogenetic tree reconstruction
The rbcL sequences were aligned using MUSCLE v3.8.31 with the
default settings (Edgar, 2004) followed by manual inspection using
BioEdit v7.2.5 (Hall, 1999). Phylogenetic analyses were conducted
using two different methods to produce a maximum-likelihood
(ML) tree and an ultrametric Bayesian tree. A nonultrametric ML
phylogeny was inferred under the best-fit nucleotide substitution
model (GTR + G+I; according to the lowest Bayesian information
criterion) with 1,000 boot strap replicates using MEGA v6.0 (Tamura
et al., 2013). An ultrametric Bayesian phylogeny was estimated
using MrBayes v3.2.2 (Ronquist et al., 2012), assuming GTR + G+I
(which was determined to be the best-fit model using MEGA) and
an Independent Gamma Rate relaxed clock (Lepage et al., 2007).
Four MCMC chains (one hot and three cold) were run for 200 million
generations, sampling every 20,000 generations and discarding the
first 80% of the posterior samples such that the average standard
deviation of split frequency was less than 0.01. A majority-rule con-
sensus tree was summarized from the MCMC trees with posterior
probabilities as support values using Mesquite v2.75 (Maddison &
Maddison, 2011). The ML tree was taken as input for the PTP species
delimitation method and the Bayesian consensus tree for the GMYC
method (see below).
2.5 | Species delimitation
DNA-based algorithmic species delimitation methods are an impor-
tant tool to differentiate species within morphologically indistin-
guishable taxonomic groups (reviewed in Leliaert et al., 2014). Several
studies have raised concerns about the accuracy of inferring species
boundaries using single-locus data (e.g., Knowles & Carstens, 2007;
Dupuis et al., 2012). To minimize potential false positives, it is ad-
vised to infer mOTUs using multiple species delimitation methods
and then to t ake the most conservative result, which has the fewest
mOTUs (Carstens et al., 2013). After determining mOTUs, the non-
freshwater taxa were excluded from downstream analyses.
For freshwater red algae, rbcL is the only marker with abundantly
available sequence data. We estimated mOTUs based on rbcL se-
quences using three different methods: automated barcode gap
discovery (ABGD), generalized mixed Yule-coalescent (GMYC), and
Poisson tree process (PTP). The distance-based method, ABGD,
identifies mOTUs by finding the threshold between intraspecific
genetic distances and interspecific genetic distances. The threshold
was determined based on the distribution of the pair wise genetic
distance between any two given sequences (corrected under the
Kimura 2 model). We employed the ABGD online tool (https://bioin
fo.mnhn.fr/abi/publi c/abgd/) using the default settings (Puillandre
et al., 2012). Next, we inferred mOTUs using two coalescent-based
methods, GMYC and PTP. GMYC determines the point of transition
from interspecific branching (a pure birth process) to intraspecific
branching (a neutral coalescent process) on a clock-calibrated ultra-
metric tree (Pons et al., 2006). For the GMYC analysis, the ultramet-
ric tree was imported into Splits (Pons et al., 2006) in R, following the
procedure described in Pons et al. (2006). PTP determines mOTUs
by detecting the threshold between the intraspecific branching rate
and the interspecific branching rate (Zhang et al., 2013). For the PTP
ana lysis, the ML tr ee was im ported into th e PT P web ser ver (ht tp s://
speci es.h-its.org/).
2.6 | Rarefaction analysis
To evaluate sampling effor t, we estimated the projected maximum
number of mOTUs and samp le size-based completeness using iNEX T
(iNterpolation and EXTrapolation; Hsieh et al., 2014) in R. The num-
ber of samples (i.e., the sample size, or more specifically the number
of rbcL seq uen ce s in this stu dy) nee ded to de te c t the max imum nu m-
ber of mOTUs were estimated by a rarefaction analysis. Sample size-
based completeness is defined as the number of samples collected
divided by the number of samples needed to capture the maximum
number of mOTUs.

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ZHAN et A l.
2.7 | Identification of introduced species
Low genetic diversity and large geographical distribution are sug-
gested to be informative criteria to identify potential introduced
taxa in the red algae (e.g., Necchi et al., 2013; Díaz-Tapia et al., 2018;
Johnston et al., 2018). We estimated the genetic diversity and ge-
ographical range of each mOTU. Two indices of genetic variation,
haplotype diversity (Hd) and nucleotide diversity (π), were computed
using DnaSP v6 (Rozas et al., 2017). Before calculating these indices,
the multiple sequence alignment was trimmed on both ends, where
there were overhanging bases (or missing data). Also, we computed
the maximum distance between any two field locations for a mOTU
as a measure of the geographical range of the mOTU. These data
were obtained for the five aquarium mOTUs with sufficient se-
quence data (at least three aquarium samples and three field sam-
ples in Taiwan) to estimate local (i.e., in Taiwan) and global genetic
variation. We considered mOTUs with no local genetic variation
(Hd = 0 and π = 0) and large geographical range (at least 10,00 0 km)
as likely to be introduced through the aquarium trade in Taiwan.
Additionally, for the introduced mOTUs with enough sampling,
their rbcL haplotype networks were inferred using PopArt v1.7
(Leigh & Bryant, 2015). Haplotype networks have been utilized to
identif y the potential geographical source(s) of introductions (e.g.,
Kato et al., 2009). Populations with higher genetic diversity may be
the source of introduced species, which often undergo a genetic bot-
tleneck (e.g., Allendorf & Lundquist, 2003; Roman & Darling, 2007).
2.8 | Trade data
There are no customs records for freshwater red algae, as they are
not traded. We sought for trade data on ornamental aquatic plants
and freshwater fish instead, because freshwater red algae hitchhike
on those traded organisms. We found no customs records on orna-
mental aquatic plants imported to and exported from Taiwan in the
Taiwan Customs database. But we found import and expor t data for
ornamental freshwater fish between 2013 and 2017 (under the HS
code “0301110090 Other Ornamental Fish, Freshwater” from the
Customs Administration, Ministry of Finance, Taiwan; https://portal.
sw.nat.gov.tw/APGA/GA03).
3 | RESULTS
ABGD, PTP, and GMYC inferred 27, 28, and 28 mOTUs in the 213
samples collected for this study, respec tively (Figure S3). We took
the most conser vative estimate of 27 mOTUs (26 are freshwa-
ter taxa and one is brackish) inferred using ABGD (initial partition
with prior maximal distance, p = 7.74 × 10 –3; distance K80 Kimura,
MinSlope = 1.5) . Th e AB GD analysis in di cate d that glo ba ll y there are
170 mOTUs of freshwater red macroalgae, of which 26 (~15%) are
found in Taiwan. A rarefaction analysis showed that excellent sam-
pling effort was achieved to detect mOTUs (95% and 96% sample
size-based completeness for the field and aquarium samples, respec-
tively; Figure 3).
Of the 26 freshwater mOTUs, 13 (50%) are found in the field
only, four (~15%) in aquaria only, and nine (~35%) mOTUs in both the
field and aquaria (Figure 3a). We have enough samples (at least three
from the field and aquaria) for only five of the mOTUs to estimate
nucleotide diversity and haplotype diversity (Table 1). Therefore, we
identified potential introduced taxa among the five mOTUs based on
genetic and geographical dat a. Three of these five mOTUs (Kumanoa
mahlacensis m OTU 067, Montangnia macrospora mOTU120, and
Thorea hispida mOTU122) exhibit no local genetic variation (i.e., in
Taiwan) in the field and aquarium samples (Table 1) and are found
across large geographical distances (i.e., across continents). We fur-
ther examined these three mOTUs as taxa that might have been in-
troduced into Taiwan.
FIGURE 3 Distribution of freshwater red algal mOTUs (a) and
propor tion of chantransia (versus gametophyte) (b) found in the
field and aquarium samples from Taiwan. Only the specimens of
the taxa of Batrachospermales and Thoreales were counted for
panel (b). The observed number of mOTUs, sampling effort, and
the number of maximum mOTUs projected using iNEXT are shown
in parentheses at the top of panel (a). The type of tank where the
aquarium samples were collected is indicated in panel (b). Taxa
exhibiting no genetic variation in either the field or aquarium
samples are marked by the hash sign. The asterisks indicate a
significant difference as per Fisher's exact test (p < .05)

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For M. macrospora (which has decent global sampling), a haplo-
type network was reconstructed to identif y the potential source
population(s) of this introduced alga (Figure 4). Eight distinct rbcL
haplotypes were recovered (designated H1 to H8). All eight haplo-
types (H1 to H8) were found in South America (specifically, Bolivia,
French Guiana, and Brazil), whereas only two haplotypes (H1 and
H2) were found in East Asia. H1 appears to be the most geograph-
ically widespread haplotype (at least based on the present sam-
pling), and it is the only haplotype found in the field and in aquaria in
Taiwan. Interestingly, H2 was not found in Taiwan but was found in
Hong Kong, Thailand, and South America. These data suggest that
South America might have been the source of M. macrospora (high
haplotype diversity) and East Asia (with sampling densely concen-
trated in Taiwan in this study) might have been the sink (low haplo-
type diversity).
Of the freshwater red algae we found in aquarium shops, only
the algae of Batrachospermales (Kumanoa, Sheathia, and Virescentia
in our data) and Thoreales (Nemalinopsis and Thorea in our data)
have morphologically distinct alternating life stages. We observed
that the relative proportion of gametophyte and chantransia (which
were identified morphologically) is different between the field and
aquarium samples (p = 6.34 × 10 –12 , Fisher's exact test; Figure 3b),
indicating that chantransia (the asexual stage) is the more frequently
occurring reproductive form in aquaria. Chantransia do not seem
to have substrate preference between aquatic plant tanks and fish
tanks (p = .86, Fisher 's ex act test; Figure 3b). However, we observe d
a higher proportion of chantransia specimens in aquatic plant tanks
than fish t anks (Figure 3b), suggesting that chantransia might hitch-
hike more often on ornamental aquatic plant s than animals. Also, we
observed that in both types of tanks, chantransia seem to grow pref-
erentially on inanimate substrates (e.g., plastic materials or rocks)
rather than aquatic plants or animals (Figure 3b; Table S1).
4 | DISCUSSION
The diversity of freshwater red algae in the aquarium trade has been
largely unexplored, because the algae are easy to miss as minuscule
spores and are often morphologically indistinguishable (i.e., cryp-
tic). Collectively, only 10 species of freshwater red macroalgae have
been documented in Taiwan on the basis of molecular or morpholog-
ical evidence (Wu, 1999, 2001; Liu et al., 2004; Chou & Wang, 20 06;
Vis et al., 2010; Chou et al., 2014; Chou et al., 2015). Through our
broad DNA barcode-based survey of field sites and aquarium shops
across Taiwan, we found 26 mOTUs of freshwater red macroalgae
(13 mOTUs in the field only, four mOTUs in aquaria only, and nine
mOTUs in the field and aquaria). To the extent that the mOTUs cor-
respond to distinct species, this result indicates that the biodiversity
of freshwater red macroalgae in Taiwan is substantially richer than
previously reported.
In this study, we found that some freshwater red algae might
have been introduced via the aquarium trade, in line with previ-
ous observations (e.g., Stoyneva et al., 2006; Kato et al., 2009).
TABLE 1 Maximum pairwise geographical distance, haplotype diversity (Hd), and nucleotide diversity (π) of five molecular operational taxonomic units (mOTUs) found in the aquarium
shops sur veyed in Taiwan in this study. These taxa have been reported in East Asia, Europe, Oceania, North America, and South America. The potential introduced taxa are bold and
underlined. Hd and π were estimated for the aquarium samples and the field samples in Taiwan separately, together, and in combination with the global field samples from NCBI GenBank
Tax a
Distance
(km) Distributional region(s)
Aquarium (Taiwan) Field (Taiwan) Aquarium + field (Taiwan)
Aquarium + field
(world, including Taiwan)
nHda πnHda πnHda πnHda π
C. caeruleus
(mOTU238)
25,102 E. Asia, Oceania, Europe, N.
America, S. America
30.000 (1) 0.00000 11 0.182 (2) 0.00171 14 0.473 (3) 0.00219 72 0.487 (4) 0.0 0167
M. macrospora
(mOTU120)
24,792 E. Asia, S. America 70.000 (1) 0.00000 18 0.000 (1) 0.00000 25 0.000 (1) 0.00000 63 0.620 (8) 0.01225
K. mahlacensis
(mOTU067)
16,394 E. Asia, Europe,
N. America
60.000 (1) 0.00000 50.000 (1) 0.00000 11 0.000 (1) 0.00000 16 0.125 (2) 0.00293
T. hispida
(mOTU122)
16,186 E. Asia, Europe,
N. America
14 0.000 (1) 0.00000 70.000 (1) 0.00000 21 0.000 (1) 0.00000 38 0.149 (2) 0.00035
S. dispersa
(mOTU010)
10,175 E. Asia, N. America 13 0.682 (4) 0.00633 26 0.538 (3) 0.0 0365 38 0.572 (5) 0.00442 87 0.742 (7 ) 0.00344
aThe number of distinct haplotypes is parenthesized.

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ZHAN et A l.
Using the amount of genetic variation and the ex tent of geograph-
ical distribution as criteria, we identified three mOTUs (Kumanoa
mahlacensis, Montangnia macrospora, and Thorea hispida) as poten-
tial introduced species among the 13 mOTUs found in aquaria in
Taiwan. The three mOTUs were also found in the field samples,
suggesting successful introduction. M. macrospora has been re-
ported to be a non-native species in a eutrophic artificial dam in
Okinawa, Japan (Kato et al., 2009). T. hispida has been reported
to be cosmopolitan with low genetic diversity, possibly partly due
to the aquarium trade (Johnston et al., 2018). This epizoic alga
can hitchhike on rusty crayfish (Orconectes rusticus), which is a
common invasive species in Asia (Fuelling et al., 2012). As far as
we know, K. mahlacensis is not known as an introduced species
elsewhere in the world. On the basis of our data, we hypothesize
that K. mahlacensis has been introduced to Taiwan via the global
aquarium trade.
Routes of introduction can be inferred based on genetic and
geographic occurrence data (e.g., Bonet t et al., 20 07; Muirhead
et al., 2008; Le Roux et al., 2011). We illustrate this using M . mac-
rospora. Kato et al. (2009) have proposed that M. macrospora was
introduced by the aquarium trade between South America and
Okinawa (Japan). Later, this alga was reportedly found in the field
in Taiwan and Malaysia (Chou et al., 2014; Johnston et al., 2014). In
our study, M. macrospora was found in the aquarium shops surveyed
in Taiwan, Okinawa (Japan), Hong Kong, and Thailand. We observed
that the nucleotide diversity and haplotype diversity of the rbcL se-
quences from the South America samples of M. macrospora (n = 25,
π = 0.02183, Hd = 0.820, eight haplot ypes; from Bolivia, Brazil, and
French Guiana) are higher than the nucleotide diversit y and hap-
lotype diversit y of the rbcL sequences from the East Asia samples
(n = 38, π = 0.00004, Hd = 0.149, two haplotypes; from Taiwan,
Thailand, Okinawa in Japan, and Malaysia). Only two haplotypes
(H1 and H2) were found in East Asia (Figure 4). Interestingly, the
trade data of ornamental fish in Taiwan indicate that there is high
import activity to Taiwan from South America (Figure S4; Table S2).
This observation lends plausibility to an introduction route between
the two regions. Taken together, these data suggest that M. mac-
rospora might have been introduced to East Asia (possibly more than
once, given that there are two haplot ypes in East Asia) from South
America (but the country of origin is unknown due to limited field
sampling) and then it might have spread fur ther among the regions in
East Asia through the aquarium trade. Plausible introduction routes
for the other two cases (K. mahlacensis and T. hispida), however, are
less clear because of insufficient sampling from field populations
(Table 1).
In addition to potential introduced taxa, we identified cryp-
tic ones. Our analysis uncovered three cr yptic taxa of Thorea
(mOTU128, mOTU129, and mOTU130) (Figure 3; Figure S3). These
cryptic taxa may be novel species that have not been described tax-
onomically. Interestingly, two of them (mOTU128 and mOTU130)
were found to be common in the aquarium shops in Taiwan but not
in the field anywhere in the world (at least based on the current sam-
pling of rbcL sequences) (Figure 3; Figure S3). These mOTUs are ex-
amples of cryptic taxa that have gone unnoticed under the current
morphology-based biodiversity monitoring programs. For example,
chantransia (the sporophytic stage) of freshwater red macroalgae
are minute hitchhikers and morphologically alike. Such cryptic taxa
have the potential to become invasive. Therefore, it is impor tant to
incorporate better tools, such as DNA barcodes, into biodiversity
monitoring approaches.
FIGURE 4 Haplotype network of Montagnia macrospora, a potential introduced species identified in this study. A network of eight
distinc t haplot ypes (H1 to H8) was inferred by TCS. The hatch marks on the links between the haplotypes represent mutational steps. The
asterisks indicate the aquarium samples. The field and aquarium samples from East Asia contain only H1 and H2 (low haplotype diversity),
whereas all eight haplotypes were found in the field samples from South America (high haplotype diversit y). Abbreviations: BL, Bolivia;
BR, Brazil; FG, French Guiana; HK, Hong Kong; MY, Malaysia; OK, Okinawa; TH, Thailand; and TW, Taiwan [Colour figure can be viewed at
wileyonlinelibrary.com]

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Here, we found that nine out of 22 freshwater red algae (~41%)
from the field also occurred in the aquaria. This observation led us
to consider the possibility that freshwater red algae may be acci-
dentally harbored in the aquarium trade. Also, some of the species
may be locally threatened, possibly due to anthropogenic distur-
bances (e.g., Nemalionopsis shawii (previously N. tortuosa) and Thorea
gaudichaudii in Japan; Brodie, Andersen, Kawachi, & Millar, 20 06).
Thus, the aquarium trade may act as an ex situ preser vation site (or
a reservoir) for freshwater red algae, which are being threatened by
water pollution caused by urbanization or industrialization (Sheath &
Hambrook, 1990; Sheath & Vis, 2015).
The criteria adopted in this study to identify potential introduced
taxa may be too stric t. Introduction of a species into a non-native
habitat can cause a genetic bottleneck that results in a drastic re-
duction of genetic diversity; however, multiple introductions of a
species into the same non-native habitat can lead to appreciable
levels of genetic diversity (reviewed in Wilson et al., 2009). Thus, po-
tential cases of introduction elsewhere (or in Taiwan) may be missed
under our stringent criteria. One example is Composopogon caeru-
leus, which is frequently found in aquaria (e.g., Stoyneva et al., 2006;
Levente & Bud, 2010; Carlile & Sherwood, 2013; Necchi et al., 2013).
This alga has been reported as a tropical invader in Belgium via the
aquarium trade (Stoyneva et al., 2006). However, we ruled out this
alga as an introduced species in Taiwan, because it did not meet our
criterion of a stark lack of local genetic diversity (Table 1).
For this study, we selected rbcL as a marker to track potential
introductions because the sequence data for this gene are abun-
dantly available and well represented geographically for freshwater
red algae compared to other nuclear, mitochondrial, and plastid loci.
The rbcL sequences of the specimens sampled worldwide enabled
us to identify tentative cases of freshwater red algae introduced to
Taiwan from distant regions. In future investigations, it may be fruit-
ful to explore the utility of alte rnative marker genes (e.g., rp oC1; Zha n
et al., 2020) and population-level markers (e.g., microsatellite loci;
Kinziger et al., 2011) to unveil candidate algal introductions along-
side rbcL. Population-level markers would help to better evaluate
differences in genetic diversity between an introduced population
and its potential source population(s) (e.g., Muirhead et al., 2008),
thereby refining our criteria of potential introduced algae. Presently,
a disadvantage of using these marker loci is their geographically re-
stricted sampling, which limits our capacity to detect long-distance
introdu ctions (e.g., between Taiwan and South America). Enough se-
quence data for those marker loci (broader geographical sampling)
need to be obtained before employing the marker loci to detect po-
tential instances of introduced freshwater red macroalgae.
Here, we examined rbcL sequences for genetic evidence that
freshwater red macroalgae are dispersed via the global aquar-
ium trade. Besides the aquarium trade, waterfowl may ser ve as
another mechanism of short- or long-distance dispersal of fresh-
water red algae. Waterfowl are thought to be active dispersers of
non-native aquatic organisms, including algae (reviewed in Reynolds
et al., 2015). Short-distance dispersal of freshwater red algae (such
as organisms of Batrachospermales) by waterfowl is plausible. We
found one case of potential introduction via short-distance disper-
sal—N. shawii, which is found only in East Asia based on the pres-
ent sampling. However, long-distance, transoceanic dispersal of the
freshwater algae by waterfowl is presumably unlikely, because these
algae obviously cannot tolerate saltwater or long-term desiccation.
Taxa of M. macrospora and T. hispidia found in this study are some
examples of freshwater algae dispersed over the oceans. Our ge-
netic diversity estimates suggest that M. macrospora has been intro-
duced from South America to East Asia, and indicated that T. hispidia
is distributed in East Asia, Europe, and Nor th America. Hence, for
these two algae, the aquarium trade is a more probable mechanism
of dispersal than waterfowl. Moreover, another possible mechanism
to introduce non-native freshwater algae is ballast water discharge,
which is known to wreck environmental havoc by spreading inva-
sive species (e.g., zebra mussel). Manny et al. (1991) speculated that
C. caeruleus, a freshwater red alga, might be transported via ballast
water discharge, but so far it remains to be a speculation. Although
ballast water discharge is an unlikely mechanism to introduce fresh-
water algae into Taiwan (an island surrounded by seawater), it may
transport freshwater algae between distant locations connected
by freshwater bodies, such as lakes and rivers, in other parts of the
world.
DNA barcoding of individual specimens, as performed in this
study, can be labor-intensive and time-consuming. This approach
provides a restricted view of the biodiversit y in an ecosystem. A
powerful alternative is environmental DNA metabarcoding enabled
by high-throughput sequencing. Recently, this approach has been
applied for large-scale biodiversity monitoring of various ecosys-
tems (reviewed in Thomsen & Willerslev, 2015). We envisage that
environmental DNA metabarcoding can accelerate the identifica-
tion of introduced, cr yptic, and possibly threatened taxa of fresh-
water red algae in the field and aquaria, thereby expanding our
knowledge of the biodiversity of these algae in the wild and artificial
environments.
Hitchhiking freshwater red algae have gone overlooked over the
past decades. We sur veyed the biodiversity of freshwater red mac-
roalgae in the field and aquaria across Taiwan and in nearby regions.
We identified potential introductions of freshwater red macroalgae
into Taiwan through the global aquarium trade. We anticipate that
our data will be a taxonomic resource for future large-scale moni-
toring programs that utilize DNA barcoding and/or environmental
DNA metabarcoding, especially around large urban centers where
aquarium trade activit y is relatively high (Strechker et al., 2011).
Overlooked hitchhikers, such as freshwater red macroalgae, are
not regulated by CITES (Convention on International Trade in
Endangered Species of Wild Fauna and Flora), hampering the pre-
vention of their spread via the aquarium trade. It is important to
educate aquarists and the public about the proper disposal of aquar-
ium waste (e.g., putting it in solid waste for compost, microwaving/
freezing it prior to the waste disposal, or treating it with chemicals),
as suggested by Padilla and Williams (2004), Patoka et al. (2016),
and Vranken et al. (2018). Furthermore, detailed studies about the
potential ecological and social concerns of algal introductions (e.g.,

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ZHAN et A l.
the homogenization of global taxonomic diversity, loss of local biodi-
versity, and potential transfer of pests or pathogens) are needed to
determine whether or not hitchhikers should be actively controlled
or eradicated in the aquarium trade.
ACKNOWLEDGEMENTS
We thank Dr. Jaruwan Mayakun (Prince of Songkla University) and
Dr. Paul Geraldino (University of San Carlos) for helping to collect
the specimens from Thailand and the Philippines. We also thank
Prof. Sarah P. Otto (University of British Columbia) for critically re-
viewing the manuscript. SHZ was supported by the UBC Four Year
Doctoral Fellowship and CIHR Doctoral Research Award. This work
was supported by grants from Ministr y of Science and Technolog y,
Taiwan to SLL (MOST108-2621-B-029-005-MY3).
AUTHOR CONTRIBUTIONS
S.H. Z. and S.L.L. conceived the idea. S.L.L. and T.Y.H. designed the
study. S.L.L., S.H. Z., T.Y.H., and S. S. performed specimen collection
and laboratory experiment s. S.L .L., L.Y., and T.C.K. analyzed the
data. S.H.Z. and S.L.L. wrote the manuscript. All authors discussed
the results and reviewed the manuscript.
DATA AVAIL ABI LIT Y S TATEM ENT
The rbcL sequences obtained in this study are deposited in NCBI
GenBank under the following accessions: MH835465-MH835677.
Table S1 is also available online in Dryad: doi:https://doi.org/10.5061/
dryad.3n5tb 2rf8.
ORCID
Shing Hei Zhan https://orcid.org/0000-0002-6351-8873
Shao-Lun Liu https://orcid.org/0000-0002-4936-0459
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SUPPORTING INFORMATION
Additional supporting information may be found online in the
Supporting Information section.
How to cite this article: Zhan SH, Hsieh T-Y, Yeh L-W, Kuo T-C,
Suda S, Liu S-L . Hidden introductions of freshwater red algae
via the aquarium trade exposed by DNA barcodes.
Environmental DNA. 2021;3:481–491. https://doi.org/10.1002/
edn3.139