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Rev. Biol. Trop. (Int. J. Trop. Biol. ISSN-0034-7744) Vol. 64 (3): 1311-1331, September 2016

Brachiopods, sipunculans, enteropneusts and metals

from two estuarine tidal flats, Pacific, Costa Rica

José A. Vargas

1,2

, Jenaro Acuña-González

1,3,4

, Fiorella Vásquez

& Jeffrey A. Sibaja-Cordero

1,2

1. Centro de Investigación en Ciencias del Mar y Limnología (CIMAR), Universidad de Costa Rica, 11501-2060, San

José, Costa Rica; jose.vargas@ucr.ac.cr, jeffrey.sibaja@ucr.ac.cr

2. Escuela de Biología, Universidad de Costa Rica, 11501-2060, San José, Costa Rica; f.vasquezfallas@gmail.com

3. Centro de Investigación en Contaminación Ambiental (CICA), Universidad de Costa Rica, 11501-2060, San José,

Costa Rica; jenaro.acuna@ucr.ac.cr

4. Escuela de Química, Universidad de Costa Rica, 11501-2060, San José, Costa Rica

Received 15-II-2016. Corrected 16-V-2016. Accepted 31-V-2016.

Abstract: Reports on the abundances and on metal concentrations in intertidal estuarine invertebrates from the

Eastern Tropical Pacific are rare. Thus, the objectives of this report are to make accessible data on the abundances

(1984-1987, 49 dates; 2013, 12 dates) of sipunculans, brachiopods and hemichordates from a sand-mud flat; and on

trace metals (1996, 2000) and abundances (2015, 3 dates) of sipunculans and brachiopods at a sand flat in the Gulf

of Nicoya estuary (10

N-85

W). Cores (17.7 cm

) were collected at the sand-mud flat, and quadrats (0.2 m

) at

the sand flat. The flats contrasted in their sand (65 % vs 90 %) and silt+clay (31.5 % vs 5.6 %) contents. At the

sand-mud flat (1984-87: 1.83 m

) the sipunculans were represented by 13 individuals, the brachiopods by 129 and

the acorn worms by 185, with estimated maximum densities of: 5.7, 29, and 40 ind./m

, respectively. Trace metal

(Fe, Mn, Ni, Cr, Cd, Zn, and Pb) analysis (Atomic Absorption Spectrometry) were conducted in specimens of

Sipunculus nudus (Sipuncula) and Glottidia audebarti (Brachiopoda). Maximum mean concentrations in S. nudus

were: For non-depurated worms, Fe (16.0 mg/g dw) > Mn (165 µg/g dw) > Zn (81 µg/g dw) > Cu (26 µg/g dw)

> Cr (11 µg/g dw) > Ni (10.4 µg/g dw) > Pb (9.3 µg/g dw) > Cd (1.2 µg/g dw). For 72 hour depurated worms:

Fe (5.0 mg/g dw) > Mn (61 µg/g dw) > Zn (39 µg/g dw) > Cu (24 µg/g dw) > Ni (8.4 µg/g dw) > Pb (2.7 µg/g

dw) > Cd (0.62 µg/g dw). For G. audebarti: Fe (1.6 mg/g dw-soft parts) > Zn (123.5 µg/g dw-soft parts) > Cu

(31.4 µg/g dw-pedicles) > Pb (21.0 µg/g dw-shells) > Cd (5.2 µg/g dw-soft parts) > Cr (4.7 µg/g dw-shells). For

sediments; Fe (46 mg/g dw) > Mn (41.3 µg/g dw) > Zn (63 µg/g dw) > Cu (36.2 µg/g dw) > Cr (31.5 µg/g dw)

> Pb (21.1 µg/g dw) > Ni (16.1 µg/g dw) > Cd (1.1 µg/g dw). These concentrations were expected for a non-

industrialized estuary. At the sand flat (Area sampled: 10.6 m

) 76 individuals of G. audebarti, 112 of G. albida,

and 366 of S. nudus were collected in 2015, with estimated maximum densities of: 7.1, 10.5, and 31 ind./m

respectively. Densities of G. audebarti and G. albida were relatively low, while those of S. nudus were relatively

high when compared with other reports. The shell lenght of G. audebarti ranged from 9.0 mm to 38.0 mm and from

6.0 mm to 29.0 mm for G. albida. These ranges were within those found for these lingulides elsewhere. The mean

length of S. nudus was 41 mm and the maximum weight was 1.6 g, which are small. No brachiopods were found at

the sand-mud flat in 2013, nor enteropneusts at the sand flat in 2015. G. audebarti had a relatively stable presence,

while G. albida almost vanished from the samples at the end of 2015. The spatial distributions of the three inverte-

brates were found aggregated at both intertidal flats. Strong ENSO warming events during 1983 and 2015, and red

tides in 1985 may have influenced the abundances. Rev. Biol. Trop. 64 (3): 1311-1331. Epub 2016 September 01.

Key words: Glottidia, Sipunculus, Apionsoma, acorn worms, tropical benthos, infauna, pollution, depuration.

From 1979 to 1983 benthic surveys were

performed in the Gulf of Nicoya estuary,

Pacific coast of Costa Rica, to evaluate its

biodiversity and conservation status (Vargas

1995, 2016). These surveys were followed by a

three-year study (1984-1987) of the community

of macro-invertebrates from the Punta Morales

intertidal sand-mud flat in the upper estuary

(Vargas, 1987, 1988, 1989, 1996). Other pub-

lications followed focusing on the abundances

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of the main taxonomic groups, like cephalo-

chordates (Vargas & Dean, 2010), echinoderms

(Vargas & Solano, 2011), mollusks (Vargas-

Zamora & Sibaja-Cordero, 2011), crustaceans

(Vargas-Zamora, Sibaja-Cordero, & Vargas-Cas-

tillo, 2012), and polychaetes (Vargas-Zamora,

Sibaja-Cordero, Dean, & Solano-Ulate, 2015).

Vargas (1987, 1988) also listed the presence

of peanut worms (Sipuncula), Glottidia aude-

barti (Broderip, 1835) lamp shells (Brachiop-

oda) and acorn worms (Enteropneusta). Emig

and Vargas (1990) reported the presence of both

G. audebarti and G. albida (Hinds, 1844) in

tidal flats from the Gulf of Nicoya. Comparative

studies on G. audebarti by Lecuyer, Grandjean

and Emig (1996) and by Kowalewski, Dyreson,

Marcot, Vargas, Flessa, and Hallman (1997)

included specimens from the Gulf of Nicoya.

The peanut worms were identified by Cutler,

Cutler and Vargas (1992) as Sipunculus nudus

Linnaeus, 1766 and Apionsoma trichocephalus

Sluiter, 1902. Recent reviews of the species

of brachiopods and sipunculans reported from

Costa Rica were conducted by Emig (2009) and

Vargas and Dean (2009), respectively. The acorn

worms remain as yet unidentified.

At the nearby Cocorocas sand flat, a pre-

liminary evaluation of the presence of metals

in S. nudus and G. audebarti was presented

by Vargas and Abdullah (1997) and the final

results are included herein. A comparison of

the Cocorocas macrofauna with that of similar

flats in other latitudes was made by Dittmann

and Vargas (2001) and by Dittmann (2002). At

Cocorocas the two species of brachiopods and

S. nudus coexist and are important components

of the macrofauna.

As a follow up of these surveys, an assess-

ment of pollutants in sediments and invertebrates

was started in 2000 at four coastal embayments

of Costa Rica, including the Gulf of Nicoya.

Concentrations of petroleum hydrocarbons in

sea water, and trace metals and Poly-Chlo-

rinated-Byphenyls (PCBs) in sediments were

reported by Acuña-González, Vargas-Zamora,

Gómez-Ramírez and García-Céspedes (2004),

García-Céspedes, Acuña-González and Vargas-

Zamora (2004) and Spongberg (2004), respectively.

In addition, coliform bacteria and beach lit-

ter were studied by García, Acuña-González,

Vargas-Zamora and García-Céspedes (2006).

The presence of imposex in Acanthais brev-

identata snails from the Gulf was informed by

Gravel, Johanning, McLachlan, Vargas, and

Oberdörster (2006) and concentrations of PCBs

in S. nudus and other sipunculans were reported

by Spongberg (2006). The abundance and spa-

tial distribution of the giant onuphid polychaete

worm Americonuphis reesei in sand flats from

the upper Gulf of Nicoya was described by Rojas

and Vargas (2008). The presence of Pharma-

ceuticals and Personal Care Products (PCCP) in

rivers draining into the Gulf of Nicoya and other

coastal areas was evaluated by Spongberg et al.

(2011). More recently, Vargas, Acuña-González,

Gómez and Molina (2015) summarized data on

trace metals from snails and clams collected at

the four coastal sites, including the razor clam

Tagelus affinis from the Cocorocas sand flat.

No recent published data is available on

the abundances of brachiopods and sipunculans

from the Gulf of Nicoya estuary. Reports are

also rare on the abundances of these groups and

acorn worms for the Eastern Tropical Pacific

region. Data on trace metals in sipunculans

and brachiopods are scarce worldwide. There-

fore, concentrations of trace metals in S. nudus

and G. audebarti from the 2000 survey are

also included herein. This information may

be crucial in studies assessing the potential

recovery of a disturbed system like the Gulf of

Nicoya estuary (Vargas, 2016). This assessment

needs information on the structure (diversity

and abundance) and function (energy flow) of

its benthic fauna, and the presence-absence of

certain groups of invertebrates may be key diag-

nostic factors as the review by Borja, Dauer and

Elliott (2010) indicates.

Thus, the objectives of this study were

to make accessible data on the abundances of

sipunculans, brachiopods and hemichordates

(1984-1987, 2013) at a sand-mud flat; and

on trace metals (1996, 2000) and abundances

(2015) of sipunculans and brachiopods at a sand

flat in the upper Gulf of Nicoya estuary.

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MATERIAL AND METHODS

Collection sites: Samples were collect-

ed at two intertidal sedimentary environments

located on the Southern shore of the Punta

Morales Peninsula, upper Gulf of Nicoya estu-

ary (10

N - 85

W), Pacific coast of Costa Rica

(Fig. 1A) during 1984-1987 and 2013 (Punta

Morales sand-mud flat) and 1996, 2000, and

2015 (Cocorocas sand flat).

The Punta Morales flat is characterized

by soft sediments. A person walking on them

quickly sinks to knee high (Fig. 1B). Accord-

ing to Vargas (1987, 1988, 1996) conspicuous

biogenic structures are tubes of the onuphid

polychaetes Diopatra ornata and Americonu-

phis reesei (Fig. 1B) and fecal mounds of hemi-

chordates. Snails like the scavenger Nassarius

luteostoma and the predator Natica unifasciata

crawl on the sediment surface year around,

while the sand dollar Encope stokessi is more

frequent during the dry seasons. At low tide the

sediments remain wet (Fig. 1B) and small tide

pools serve as refuge for crabs, shrimps, and

gobiid fishes. Several species of shore birds feed

at low tide on this fauna.

The Cocorocas sand flat is characterized

by dark-gray sandy sediments allowing a per-

son to walk and stand on them without sinking

(Fig. 1C). The flat is under the influence of

freshwater discharges from the nearby Lagarto

River that carries heavy loads of sediments dur-

ing the rainy season, and coarse sediments are

deposited on the flat. Ripple marks are observed

at low tide as well as occasional ray feeding

pits. Tubes of onuphid worms are scarce and

are mainly of D. ornata. Hemichordate fecal

mounds are absent. Snails and sand dollars are

also rare on the sediment surface. At low tide,

the sediment surface gets almost dry (Fig. 1C)

and ray feeding pits act as refuges for small

fish and crustaceans. Shore birds were rarely

seen on this flat. At both the Punta Morales

and Cocorocas flats the infauna includes the

brittle star Ophiopholis geminata. At Punta

Morales the small razor clam Tagelus bourgeoi-

sae is frequent, while at Cocorocas the larger

Fig. 1. A. Location of the Punta Morales and Cocorocas

intertidal flats. Punta Morales Peninsula, mid upper Gulf

of Nicoya estuary. Pacific, Costa Rica. (10ºN-85ºW).

1. Tempisque River, 2. Tárcoles River, 3. Lagarto River.

B. Sand mud flat. C. Sand flat. Two islets (outlined in A)

are visible in the background. Note horizontal dark band

marking high tide level on the tree islet.

Gulf of

Nicoya

Pier

Costa

Rica

10º N

85º W

Pacific

Ocean

Punta Morales

sand-mud flat

Cocorocas

sand flat

Sandy beach

Mangroves

Rocky shores

Tidal flats at low tide

0 200 m

Punta

Morales

Gulf of

Nicoya

Pacific

Ocean

10º N

85º W

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T. affinis is found and harvested occasionally

for local consumption.

In this upper region of the Gulf (Fig. 1A)

the average tidal range is 2.3 m and tides are

semi-diurnal. The estuary is under the influence

of seasonal rainfall: A rainy season occurs from

May to November, followed by a dry season

from December to April. In the upper estuary

surface water salinity varies accordingly from

about 24 psu (practical salinity units) during the

rainy season, to near 34 psu during the dry sea-

son (Voorhis, Epifanio, Maurer, Dittel, & Vargas,

1983). Primary productivity data from the Gulf

of Nicoya indicates that this estuary may have

reached the hypertrophic condition (Cloern,

Foster, & Kleckner, 2014). Runoff reaches the

Gulf by several rivers of which the Tempisque

River at the head and the Tárcoles River near

the mouth (Fig. 1A) are the more important.

Surface water temperatures are near 30

C year

around, while at low tide the exposed sediments

may reach temperatures near 40

C, particularly

during the sunny dry season. A strong El Niño

Southern Oscillation (ENSO) event influenced

the Gulf of Nicoya region during 1982-1983,

as well as during 2015. In 2015 the onset of the

rainy season was delayed several months and

rains ended in early November due to ENSO.

Sediment and specimen collections at the

Punta Morales sand-mud flat (1984-1987,

2013): The intertidal sampling protocols used

at the Punta Morales flat are outlined in Vargas

(1987, 1988). In summary, two sets of 14 cores

(17.7 cm

-15 cm deep) were collected at semi-

monthly intervals (February, 1984 to February,

1985) and one set of 14 cores at near monthly

intervals (March, 1985 - April, 1987; and Janu-

ary - December, 2013) from muddy sands dur-

ing low tide and within a 400 m

plot 20 m

apart from a sandy beach (Fig. 1A). Data from

Vargas (1989) was expanded with unpublished

information from March and April of 1987, for

a total of 49 sampling dates. Core samples were

fixed in Rose Bengal stained formalin in sea

water. Preserved cores were sieved thru a 500

micron mesh. Sorted specimens of brachio-

pods (Glottidia spp.), sipunculans (S. nudus,

A. trichocephalus), and acorn worms (Fig. 2A)

were kept in vials filled with 70 % ethanol.

Sediment cores were also collected for determi-

nations of grain size (sieve analysis according

to Gray & Elliot, 2010) and organic matter con-

tent (by combustion at 450

C), dried at 65

and kept in sealed polyester bags until analysis.

Specimen collection at Cocoracas sand

flat (1996, 2000, 2015): On June 10, 1996

individuals of S. nudus (Fig. 2B, Fig. 2C,

Fig. 2D) and of the brachiopod G. audebarti

(Fig. 2F) were collected at the Cocorocas flat

along a 100 m long transect perpendicular

to the shore and running between two islets

(Fig. 1A, Fig. 1C). Sediment clumps were

removed with a shovel to a depth of 20 cm,

broken up by hand, and brachiopods and sipun-

culans placed in acid-washed polyester bags.

At the laboratory the specimens were vacuum

dryed for international shipment. A total of 40

individuals of S. nudus, and 20 of G. audebarti

were selected to perform trace metal analyses

by means of Atomic Absorption Spectrom-

etry (AAS). Analyses were accomplished by

M. Abdullah at the Department of Biology of

the University of Oslo (Norway) on non-depu-

rated whole S. nudus, and in tissues, pedicles,

and shells of G. audearti.

On March 8th, 2000, sediment clumps

were again removed with a shovel to a depth

of 20 cm. A total of 100 specimens of S. nudus

were collected at Cocorocas and transported to

the laboratory in a cooler filled with sea water

from the site. At the laboratory the worms

were kept in cooled (20

C) sea water for two

hours, rinsed with filtered sea water, blotted

dry on a paper towel, measured, weighed, and

separated into two groups of 50 individuals

each. One group was placed in a heat-sealable,

acid-washed polyester bag and stored frozen

(-18 ºC) until analysis. The second group was

kept in a plastic container filled with filtered sea

water from the site and aerated with portable

plastic aquarium pumps to allow the worms to

remove sediments and other matter from their

digestive tracts (Fig. 2 D). Sea water, feces, and

dead organisms were discarded every 12 hours

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Fig. 2. A. Anterior regions (proboscis, collar, and part of trunk) of five Rose Bengal stained acorn worms. B. Live Sipunculus

nudus collected at the sand flat. C. S. nudus drawn from live specimen. D. S. nudus during the 72 h depuration process.

E. G. albida. Note setae bundles. F. Rose Bengal stained Glottidia audebarti. Note tips of pedicles with agglomerated

sand grains.

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and the container filled with fresh filtered sea

water. The procedure was repeated six times

for a total of 72 hours. Forty-two individuals

were alive (able to move) at the end of the

depuration period. The depurated worms were

removed by hand, blotted dry on a paper towel,

measured, weighed, and stored frozen (-18 ºC)

as above until analysis. Trace metal analyses

were made at the Research Centre on Marine

Pollution (CICA) of the University of Costa

Rica. Metal determinations (Fe, Mn, Ni, Zn,

Pb, Cd) were performed following methods

described by Vargas, Acuña-González, Gómez,

and Molina (2015). In summary: Certified

reference materials and blank tests were run.

Fe, Mn and Zn were analyzed by Flame AAS

(Perkin Elmer

3300). Cd, Ni and Pb were

analyzed by Graphite Furnace AAS (Perkin

Elmer

HGA 600). The results for iron are

expressed as mg/g dry weight (mg/g dw), and

all other concentrations are expressed in parts

per million / dry weight (μg/g dw). Sediment

samples for trace metal analysis were collected

using an acid-washed plastic corer, placed in

heat-sealable, acid-washed polyester bags, and

stored frozen (-18 ºC) until analysis.

On July 6, October 2, and December

15, 2015, peanut worms (S. nudus) and bra-

chiopods (G. audebarti and G. albida) were

collected at the Cocorocas sand flat to verify

its presence and estimate their relative abun-

dances. A wooden frame (50 x 40 cm) was

pressed on the sediment surface to mark the

sampling area (0.20 m

). A minimum of 15 (3.0

) and a maximum of 20 (4.0 m

) samples

were collected during low tide, along a 200 m

transect running between two islets (Fig. 1C)

and perpendicular to the shoreline. Each sam-

ple was collected 10 m apart from each other.

Sampling was scheduled to coincide with very

low tides near noon time. Sediment clumps

were removed with a shovel to a depth of 20

cm, broken up by hand, and brachiopods and

sipunculans placed in plastic bags with sea

water from nearby tide pools. Brachiopod

specimens from each quadrat were split in

two groups: those with cream-white shells

(G. albida, Fig. 2E) and those with dark-green

coloration on the anterior region (G. audebarti,

Fig. 2F). Brachiopods were blotted dry on

paper towel and shell length (center of shell tip

to pedicle attachment) measured to the nearest

0.5 mm. Final preservation of organisms was

in 70 % ethanol. Specimens of S. nudus were

also preserved in 70 % ethanol for 24 hours

and quickly blotted dry on a paper towel and

weighed. Samples for grain size analysis and

organic matter content were collected by hand

from sediment removed by the shovel, dried

at 65

C, and kept in sealed polyester bags as

described before.

RESULTS

Sediment compositions: The sediment

compositions of the Punta Morales (1984,

2013) sand-mud flat and of the Cocorocas

sand flat (2015) are included in Table 1. The

sand-mud flat was characterized in 1984 by

65.5 % sand (46.4 % fine sand, 15.1 % very

fine sand), the silt+clay content was 31.5 %

TABLE 1

Mean percentages of dry sediment fractions retained on sieves. Lower size (microns) limit of particles: Granules (2000),

coarse+very coarse sands (1000), medium sand (500), fine sand (250), very fine sand (63). Silt+clay: fraction washed thru

a 63 micron mesh sieve. Percent organic matter content (by combustion at 450ºC). A. Punta Morales sand-mud flat

(1984, n=10). B. Punta Morales sand-mud flat (2013, n=12). C. Cocorocas sand flat (2015, n=3).

Gulf of Nicoya estuary, Pacific coast of Costa Rica

Granules Coarse+Very coarse Medium Fine Very fine Silt+clay Total Organics

A. 0.7 0.8 3.3 46.4 15.1 31.5 97.8 2.0

B. 0.8 1.1 1.8 5.6 33.0 57.6 99.9 8.0

C. 3.7 10.7 9.1 29.2 41.7 5.6 100 3.0

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and organic matter was 2 %. A change towards

finer sediments and organic enrichment was

found during the 2013 survey: 41.3 % sand

(5.6 fine sand, 33.0 % very fine sand), the silt+

clay content was 57.6 % and organic matter

was 8 %. Coarse + very course sands were

around 1 % in both surveys (Table 1 A, B). The

Cocorocas sand flat samples contained higher

percentages of sand (90.7 %) of which 29.2 %

was fine sand. Coarse + very coarse sands were

10.7 %. The silt+clay content was only 5.6 %

and organic matter was around 3 % (Table 1 C).

The 1984-1987 survey: Examples of the

acorn worms and brachiopods collected are

included in Fig. 2A, Fig. 2F. Organisms of the

three groups had many on a size range that

may represent juvenile forms. Acorn worms

in particular included many organisms around

10 mm in length (Fig. 2A). The total numbers

were: 13 sipunculans (12 ind. of S. nudus,

1 ind. of A. trichocephalus) 129 brachiopods

and 185 hemichordates (Fig. 3). The total area

sampled was 1.83 m

The abundances of these three groups at

the Punta Morales sand-mud flat for the 49

dates of the 1984-1987 survey are included

in Fig. 3. Sampling started in February 1984,

shortly after the impact of the severe 1982-

1983 ENSO high temperatures and dry condi-

tions had returned to near normal values in

the Central American region. Red tides were

frequent in the upper Gulf of Nicoya in 1985,

particularly after June (Fig. 3). The periods

from the start of sampling to about June 1984,

and from around May 1985 to October 1985

were characterized by low to zero abundances.

In spite of the fact that after March 1985 the

sampling effort was smaller in space (14 cores)

and in time (monthly), abundances were higher

during the rainy seasons. Abundance patterns

of brachiopods and acorn worms in particular

were similar since both groups were absent

early on 1984, begun to increase and reached

high numbers around the mid rainy season of

1984, became scarcer during 1985 and early

1986, and increased slightly during the dry

season of 1986-1987. The presence of a few

sipunculans was restricted to the rainy seasons

of 1984 and 1985 (Fig. 3).

Metal concentrations (1996, 2000):

Examples of the peanut worm Sipunculus

nudus are included in Fig. 2B, Fig. 2C, Fig. 2D.

The concentrations of trace metals in non-dep-

urated S. nudus, soft parts, pedicles, and shells

of Glottidia audebarti, and sediments collected

in 1996 are included in Table 2. S. nudus had

higher mean concentrations of Fe and Cr than

G. audebarti, while the brachiopod presented

higher mean concentrations of Zn and Cd

(soft parts), Pb (shells) and Cu (pedicles).

Pb concentrations were similar in organisms

and in sediments, while those of Zn and Cd

were higher in organisms than in sediments.

TABLE 2

Mean (n = 4) metal concentrations (± Standard Deviation) in non-depurated whole Sipunculus nudus (Sipuncula),

in tissues and shells of Glottidia audebarti (Brachiopoda), and in sediments. Unpublished data from Vargas, J.A.

& M.I. Abdullah, 1997, AAS (Atomic Adsorption Spectrometry, Dept. of Biology, University of Oslo, Norway).

Cocorocas intertidal sand flat. Gulf of Nicoya estuary, Pacific, Costa Rica. March, 1996.

Concentrations in μg/g dw, except for iron (mg/g dw)

Species / metal Fe Cr Zn Pb Cu Cd

S. nudus, whole

5.4 ± 0.7 11.0 ± 5.0 56.0 ± 2.0 9.3 ± 0.2 26.0 ± 2.0 1.2 ± 0.5

G. audebarti, soft parts

1.60 ± 0.07 1.5 ± 0.1 123.5 ± 7.5 2.39 ± 0.65 7.9 ± 0.2 5.20 ± 0.15

G. audebarti, pedicles

0.48 ± 0.01 2.1 ± 0.1 42.6 ± 0.1 2.4 ± 0.1 31.4 ± 0.2 0.37 ± 0.02

G. audebarti, shells

0.57 ± 0.07 4.7 ± 0.8 73.7 ± 0.1 21.0 ± 0.6 12.88 ± 0.01 3.86 ± 0.01

Sediments 25.8 ± 5.3 31.5 ± 0.5 62.6 ± 0.5 21.1 ± 0.2 36.2 ± 0.2 1.10 ± 0.02

Blanks <0.04 <0.02 0.06 <0.05 <0.02 <0.01

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0 5 10 15 20 25

Number of individuals

Feb. 22

Mar. 08

Mar. 20

Apr. 05

Apr. 17

May 03

May 16

Jun. 01

Jun. 19

Jul. 19

Jul. 31

Aug. 14

Aug. 28

Sep. 10

Sep. 27

Oct. 10

Oct. 26

Nov. 12

Nov. 24

Dec. 10

Dec. 26

Jan. 10

Jan. 24

Feb. 07

Feb. 21

Mar. 07*

Apr. 10

May 09

Jun. 06

Aug. 19

Sep. 22

Oct. 16

Nov. 15

Dec. 16

Jan. 30

Feb. 13

Mar. 13

Apr. 29

May 26

Jun. 25

Jul. 24

Aug. 21

Sep. 19

Oct. 17

Dec. 02

Jan. 19

Feb. 20

Mar. 28

Apr. 29*

1984198519861987

Dates

0 5 10 15 20

Number of individuals

A B

1/0

1/1

1/0

3/4

2/4

4/3

2/4

9/4

5/4

10/10

6/5

0/2

5/3

5/14

1/6

4/3

3/1

1/4

1/1

2/0

3/2

5/8

7/14

7/4

4/3

7/0

2/0

1/1

13/10

Enteropneusta

N = 185

Brachiopoda

N = 129

Sipuncula

S. nudus N = 12

A. trichocephalus N = 1*

Red tides

1982-1983

Strong ENSO

Fig. 3. Number of individuals per date (49 dates) of: A. Lingulide brachiopods (Brachiopoda, light grey bars) and peanut

worms (Sipuncula, black bars). B. Acorn worms (Enteropneusta, dark gray bars) found in a 400 m

sampling plot at the

Punta Morales intertidal sand-mud flat, Gulf of Nicoya estuary, Pacific coast of Costa Rica (1984-1987). Feb. 22, 1984 to

Feb. 21, 1985: Semi-monthly sampling of two sets of 14 cores per date. Numbers of acorn worms and brachiopods found on

each 14 core set are indicated on the top of the bars. Sipunculans were found on one of the sets. Mar. 07, 1985 to Apr. 29,

1987: Monthly sampling of 14 cores per date. Core area 17.7 cm

, core depth 15 cm. Mesh screen: 500 microns.

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Mean concentrations of Fe, Cu, and Cr were

higher in sediments.

The concentrations of metals in tissues of

non-depurated and depurated whole S. nudus

collected in 2000 are included in Table 3A. In

non-depurated worms mean concentrations of

Fe and Zn were higher in 2000 than in 1996,

while Pb and Cd were lower in 2000; Cu was

detected in similar concentrations for both sur-

veys (Tables 2, 3A). When median concentra-

tions of non-depurated and depurated S. nudus

are compared, these decreased for all metals

except Cd (Table 3A).

Maximum mean concentrations of met-

als in S. nudus worms found either during the

1996 or 2000 surveys, were: For non-depurated

worms; Fe (19.4 mg/g dw) > Mn (165 µg/g

dw) > Zn (81 µg/g dw) > Cu (26 µg/g dw) > Cr

(11 µg/g dw) > Ni (10.4 µg/g dw) > Pb (9.3 µg/g

dw) > Cd (1.2 µg/g dw). For depurated worms:

Fe (6.2 mg/g dw) > Mn (61 µg/g dw) > Zn

(39 µg/g dw) > Cu (24 µg/g dw) > Ni (8.4 µg/g

dw) > Pb (2.7 µg/g dw) > Cd (0.62 µg/g dw).

However, median concentrations in non-depu-

rated vs non-depurated S. nudus were not signif-

icant (Table 3A). For G. audebarti (1996 only):

Fe (1.6 mg/g dw-soft parts) > Zn (123.4 µg/g

dw-soft parts) > Cu (31.4 µg/g dw-pedicles)

> Pb (21.0 µg/g dw-shells) > Cd (5.2 µg/g

dw-soft parts) > Cr (4.7 µg/g dw-shells). For

sediments: Fe (46 mg/g dw) > Mn (41.3 µg/g

dw) > Zn (63 µg/g dw) > Cu (36.2 µg/g dw) >

Cr (31.5 µg/g dw) > Pb (21.1 µg/g dw) > Ni

(16.1 µg/g dw) > Cd (1.1 µg/g dw).

The weight on non-depurated S. nudus

ranged from 0.65 g to 1.67 g, with an average

of 0.95 g, while the weight of depurated worms

ranged from 0.61 g to 1.35 g, with a mean of

0.84 g. The weight of depurated S. nudus was

significantly lower after the depuration period

(Table 3B). There was no statistically significant

difference in length of non-depurated vs depu-

rated organisms The mean length of S. nudus

was 45 mm, with a maximum of 65 mm

(Table 3B, Fig. 2 B.C).

The 2013 survey of the sand-mud flat:

During the 2013 monthly core sampling at the

Punta Morales sand-mud flat no specimens

of brachiopods and of S. nudus were found.

However, the Sipuncula included seven indi-

viduals of A. trichocephalus (Fig. 4). The acorn

worms (Enteropneusta) were represented by

16 specimens found mainly during the rainy

season (Fig. 4).

Fig. 4. Number of individuals per date (12 dates) of

the sipunculan, Apionsoma trichocephalus (dark bars),

and acorn worms (Enteropneusta, gray bars) found in

a 400 m

sampling plot at the Punta Morales intertidal

sand-mud flat, Gulf of Nicoya estuary, Pacific coast of

Costa Rica (2013).Monthly sampling of 14 cores per

date (core area:17.7 cm

, core depth: 15 cm, 500 micron

mesh screen). No brachiopods were found in the 168

core samples. (Area: 0.297 m

)

0 1 2 3 4 5 6 7 8 9 10

Number of individuals

Dates 2013

Sipuncula

A. trichocephalus N = 7

Enteropneusta

N = 16

Jan. 15

Feb. 11

Mar. 14

Apr. 12

May 30

Jun. 24

Jul. 29

Aug. 9

Sep. 9

Oct. 10

Nov. 8

Dec. 5

The 2015 survey of the sand flat: Exam-

ples of the brachiopod G. albida collected are

included in Fig. 2E. Samples were collected at

the Cocorocas flat in July, October, and Decem-

ber of 2015, a year when high temperatures

and dry conditions in the Eastern Pacific region

were enhanced by the ENSO event (NOAA,

2016). The presence at the sand flat of bra-

chiopods (G. albida and G. audebarti) and

S. nudus worms was confirmed during sampling

conducted in 2015. These three species were

found frequently living together on a single

quadrat area (0.2 m

). Acorn worms, however,

were not found.

A total of 76 individuals of G. audebarti,

112 of G. albida and 366 of S. nudus were

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TABLE 3

A. Metal concentrations in whole soft tissues of the deposit feeder Sipunculus nudus (n = subsamples).

B. Length of body (mm) and weigth (g) of blotted-dry live non-depurated, and after 72 hour depuration of individuals of

S. nudus (n = 42). C. Metal concentrations in sediments. D. Maximum concentrations in the razor clam Tagelus affinis

from the Cocorocas sand flat (2006)*. All concentratios in µg/g dw except for iron (mg/g dw). Cocorocas sand flat, Gulf

of Nicoya estuary, Pacific coast of Costa Rica. March 8, 2000

Depurative Treatment Statistics Fe Mn Ni Zn Pb Cu Cd

Range Min. 16.1 34.4 1.17 8.44 0.60 6.42 0.17

Max. 22.5 234.6 15.57 144.0 4.67 29.1 1.64

Mean

16.0 165 10.4 81 2.8 20.7 0.63

S.D. 8.2 113 8.0 53 1.5 9.9 0.61

5 3 3 5 5 4 5

Median 16.9 226.0 14.5 76.7 2.95 23.6 0.53

Yes

Range Min. 1.55 1.23 0.15 5.55 0.23 1.86 0.06

Max. 9.21 105.7 12.75 61.1 6.39 52.7 1.00

Mean

5.0 61 8.4 39 2.7 24 0.62

S.D. 3.9 54 7.1 23 2.3 21 0.32

5 3 3 5 5 4 5

Median 6.52 77.4 12.2 43.9 2.59 21.3 0.65

Mann-Whitney U test for equal medians of depurated vs non-depurated S. nudus: Fe (p = 0.06), Mn (p = 0.38), Ni (p = 0.38),

Zn (p = 0.21), Pb (p = 0.83), Cu (p = 0.88), Cd (p = 0.83) . All p = non-significant.

Non depurated Depurated

Length Weight Length Weight

Mean 45.1 0.955 45.0 0.840

S.D. 8.28 0.260 5.02 0.160

Min 30.0 0.650 32.0 0.611

Max 65.0 1.670 60.0 1.352

Median

45.5 0.965 45.0 0.824

Mann-Whitney U test for equal weight medians: 571 (p = 0.01) significant.

Mann-Whitney U test for equal length medians: 807 (p = 0.76) non significant.

Statistics Fe Mn Ni Zn Pb

Range Min. 20.9 203.8 7.14 30.2 5.45

Max. 60.3 549.3 20.6 83.1 12.6

Mean (n = 4)

46 413 16.1 63 10.1

S.D. 17 148 6.1 23 3.2

Median 50.4 449.2 18.3 69.9 11.1

S.D. = standard deviation. n = number of samples.

Fe Mn Ni Zn

T. affinis non depurated

2 160 ± 55 255.2 ± 3.6 4.13 ± 0.09 206.7 ± 4.1

T. affinis depurated

290.3 ± 6.5 24.97 ± 0.90 1.60 ± 0.06 153.5 ± 3.1

* Data from Vargas, Acuña-González, Gómez & Molina (2015).

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Fig. 5. Arbitrary shell length groups (mm) of brachiopods: A, B, C. Glottidia audebarti. D, E, F. G. albida. Specimens

collected on July 2 (early rainy season), October 6 (mid rainy season), and December 15 (dry season) 2015. Cocorocas sand

flat, Gulf of Nicoya estuary, Pacific, Costa Rica. Vertical dashed lines = mean length of individuals.

N = 22

Mean = 22.5 mm

SD = 5.84

Range: 14 to 38 mm

Area sampled: 4.0 m

July 06 July 06

October 02

Frequency

N = 73

Mean = 12.54 mm

SD = 3.93

Range: 6.5 to 26 mm

Area sampled: 4.0 m

N = 23

Mean = 21.0 mm

SD = 5.88

Range: 11 to 30 mm

Area sampled: 3.0 m

Frequency

October 02

N = 35

Mean = 16.0 mm

SD = 5.33

Range: 6 to 29 mm

Area sampled: 3.0 m

December 15

N = 31

Mean = 21.80 mm

SD = 4.86

Range: 9 to 31.5 mm

Area sampled: 3.6 m

Lenght (mm)

0–5

6-10

11-15

16-20

21-25

26-30

31-35

36-40

41-45

46-50

December 15

N = 4

Mean = 9.75 mm

SD = 2.87

Range: 6 to 13 mm

Area sampled: 3.6 m

Frequency

Lenght (mm)

0–5

6-10

11-15

16-20

21-25

26-30

31-35

36-40

41-45

46-50

found in a pooled sample area of 10.6 m

(Fig. 5). The size range of G. audebarti was

from 9.0 mm to 38.0 mm, while for G. albida

the range was from 6.0 mm to 29.0 mm

(Fig. 5). The weight range of S. nudus was from

0.10 g to 2.55 g (Fig. 6).

Abundances (22 - 23 - 31 ind.) of G. aude-

barti were relatively stable. The smaller and

the bigger specimens were both collected in

December, and mean shell lengths (22.5 - 21.0

- 21.8 mm) were similar during the sampling

period (Fig. 5 A, Fig. 5B, Fig. 5C). On the

other hand, G. albida abundances (73 - 35 - 4

ind.) declined sharply during the period. The

smaller specimens of G. albida were found

during the three sampling dates, while the

bigger individuals were found in July and

October, and mean shell lengths increased and

then decreased (12.5 - 16.0 - 9.7 mm), Fig. 5

D, Fig. 5E, Fig. 5F. The abundance of S. nudus

was high towards the end of the rainy season.

Individuals in the ranges below 1.0 g were

more common along the sampling period, but

the increase in numbers of those above 1.0 g

was modest (Fig. 5 A, Fig. 5B, Fig. 5C). Two

individuals of the larger (approx. 200 mm)

sipunculan, Xenosiphon branchiatus were also

found in the Cocorocas samples.

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Spatial patterns: During the 1984-1987

survey the maximum numbers of individuals of

acorn worms, brachiopods and S. nudus found

in a core (17.7 cm

) were 5, 3, and 5, respec-

tively. Estimated mean densities of the three

groups were 29, 5.7 and 40 ind./m

, respec-

tively (Table 4). During the 2015 survey mean

abundances (ind./m

) for the three sampling

dates were 7.1 (G. audebarti), 10.5 (G. albida),

and 34.5 (S. nudus), respectively. The maxi-

mum numbers of G. audebarti, G. albida,

and S. nudus found in a quadrat (0.2 m

) were

11, 18 and 20, respectively. Estimated densi-

ties (ind./m

) for the date with the maximum

number of individuals of each species were:

8.6 (G. audebarti), 18.2 (G. albida) and 46.4

(S. nudus), Table 4.

DISCUSSION

The Brachiopoda and Hemichordata are

considered two separate groups each with the

category of Phylum, while the Sipuncula is

presently grouped by some authors within the

Phylum Annelida based on molecular evidence

(Brusca, Moore, & Shuster, 2016).

The lamp shells, acorn worms and pea-

nut worms include organisms that burrow in

sediments of marine and estuarine habitats

worldwide. The early information on these

groups was summarized by Hyman (1959)

who mentioned several examples of the scarce

literature available at the time for tropical

regions. More recent reviews were published

by Cutler (1994), Emig (1997, 2009) Cameron

(2005) and Vargas and Dean (2009). These

works also indicate that for tropical inter-

tidal flats the gap of information on diversity,

distribution and abundance of these benthic

organisms continues to be significant. Dit-

tmann and Vargas (2001) and Dittmann (2002)

have pointed out that among key organisms

structuring the fauna of tropical tidal flats are

N = 82

Mean = 1.01 g

SD = 0.54

Range: 0.10 to 2.25 g

Area sampled: 4.0 m

July 06

Frequency

N = 117

Mean = 0.80 g

SD = 0.41

Range: 0.19 to 2.10 g

Area sampled: 3.0 m

FrequencyFrequency

December 15

N = 167

Mean = 0.84 g

SD = 0.91

Range: 0.19 to 2.19 g

Area sampled: 3.6 m

Weight (g)

0.10 - 0.25

0.26 - 0.50

0.51 - 0.75

0.76 - 1.00

1.01 - 1.25

1.26 - 1.50

1.51 - 1.75

1.76 - 2.00

2.01 - 2.25

October 02

Fig. 6. Arbitrary weight (grams) groups for the ethanol

preserved sipunculan Sipunculus nudus: A. July 2

(early rainy season), B. October 2 (mid rainy season),

C. December 15 (dry season), 2015. Cocorocas sand flat,

Gulf of Nicoya estuary, Pacific, Costa Rica. Vertical dashed

line = mean weight of individuals.

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burrow building sipunculans, brachiopods and

enteropneusts. Their burrowing activities and

the ingestion-egestion of sediments modify the

substrate in such a way, that it becomes suitable

or unsuitable for the settlement of larvae or the

survival of juveniles of other benthic species

(Gray & Elliott, 2009).

In this study we followed Cutler (1994)

and Emig (1983, 1997) morphological criteria

for the identification of the peanut worms as

Sipunculus nudus and Apionsoma trichocepha-

lus, and the lamp shells as Glottidia audebarti

and G. albida, respectively. The acorn worms

need further taxonomic work and we were

reluctant to assign the specimens to categories

below Class level.

As its name indicates, G. albida has a

white shell while G. audebarti is character-

ized by the green color on the anterior region

of the valves. According to Dall (1921) G.

albida shells are streaked with brown, but this

color pattern was present in only two of the

larger specimens found. The type locality of G.

albida is Magdelena Bay (California, U.S.A.)

in 12 m depth sandy-mud, and for G. aude-

barti is Guayaquil Bay (Ecuador), in intertidal

sand. The presently known distribution of G.

albida is from California to Costa Rica, while

G. audebarti is found from Mexico to Ecuador

(Emig, 2009).

The lamp-shells G. auderbarti (Broderip,

1835), and G. albida (Hinds, 1844), are fil-

ter feeding lingulide inarticulate brachiopods.

Lingulides live in burrows produced by them-

selves with the aid of shell movements. Lingu-

lides are euryhaline invertebrates. The organisms

retract into the burrows during low tide and

emerge with the incoming tide to near sediment

surface and filter the water for food. The pedicle

end serves as anchor to facilitate vertical move-

ments (Emig, 1997).

The peanut worm S. nudus is a sipunculan

that has been known to scientists since Linnaeus

described this species in 1766 (Hyman, 1959).

Specimens collected around the world have

morphological features that fit the description

of S. nudus. Therefore, this species has been

considered as cosmopolitan (Cutler, 1994). As

such, its potential for use as a worldwide indi-

cator of pollution was advanced by Vargas and

Adbullah (1997). The distribution of S. nudus

as a cosmopolitan species has been questioned

based on recent molecular evidence provided

by Kawauchi and Giribet (2013) that points

to the existence of at least four geographical

lineages. However, their study did not include

specimens from Costa Rica.

This sipunculan is a prey item for birds

foraging at low tide in the Gulf of Nicoya

(Pereira, 1996). In China and other Asian coun-

tries there is a fishery that exports live S. nudus

and sells it (market name: BB worm) as fish

bait, mainly in Japan (Saito, Kawai, Umino, &

Imabayashi, 2014). The worms are also a food

item for human consumption in Asia (Saiz-

Salinas, 1993).

TABLE 4

Distribution of organisms among sampling units for the date with the maximum number of individuals (see Figs. 3, 4, 5).

Total number and date. Estimated mean density (ind./m

). A. Punta Morales (Corer: 17.7 cm

) B. Cocorocas

(Wooden frame: 2000 cm

). Intertidal flats, Gulf of Nicoya estuary, Pacific, Costa Rica

Distribution, (total number, date), mean density / m

Ind. / m

A. 1984-1987

Enteropneusta 1 0 0 0 0 2 1 0 3 0 0 5 2 0 - - - - - - (n = 14, Dec. 26, 1984) 40

Glottidia spp.

0 0 1 0 0 0 2 0 1 0 3 1 2 0 - - - - - - (n =10, Oct. 10, 1984) 29

Sipunculus nudus

0 1 0 0 1 0 0 0 0 0 0 0 0 0 - - - - - - (n = 2, Jan. 10, 1985) 5.7

B. 2015

G. audebarti

0 4 2 0 11 2 3 1 1 1 2 0 0 1 2 0 1 0 - - (n = 31, Dec. 15) 8.6

G. albida

2 1 1 0 0 10 15 1 1 0 0 2 1 18 1 0 0 5 6 9 (n = 73, Jul. 6) 18.2

S. nudus

5 11 20 13 3 16 9 13 10 2 11 11 8 7 4 4 13 7 - - (n = 167, Dec. 15) 46.4

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S. nudus burrows actively in sandy sedi-

ments feeding on them as they burrow and has

integumentary specializations for gas exchange

thru the body surface (Ruppert & Rice, 1995)

facilitating their infaunal mode of life. Sipun-

culans are able to survive drastic changes in

salinity and their lower limit may be around

18 psu (Murina, 1984). Salinities around 20 psu

are sometimes found in the upper Gulf of

Nicoya estuary during the rainy season (Voo-

rhis, Epifanio, Maurer, Dittel, & Vargas, 1983).

The acorn worms (Enteropneusta) include

about 80 species grouped into several families

separated by the structures of the gills, gonads,

liver sacs, and coelomic diverticula (Cameron,

2005). The need to observe internal struc-

tures in complete specimens makes their proper

identification difficult for the non-specialist. In

addition, benthic sampling gear (grab, corer)

normally yields fragmented specimens of these

fragile and usually long organisms. The study

by Deland, Cameron, Rao, Ritter, & Bullock

(2010) on the family Harrimaniidae of acorn

worms points out that the Eastern Pacific har-

bors many yet unknown species. In spite of

these identification difficulties, information on

the presence and abundance of acorn worms is

useful for future comparisons of the structure

(species diversity and abundance) and function-

ing (energy flow) of tidal flat systems. In spite

of the fact that data from the Punta Morales flat

was collected more than three decades ago, it

remains as the only study on the ecology of these

three groups of invertebrates for this region of

the Eastern Tropical Pacific.

Emig & Vargas (1990) pointed out that

the 102 small specimens reported by Vargas

(1987) as G. audebarti may include individuals

of both species, while the five larger speci-

mens collected the following year were of G.

audebarti. Thus, abundances of lamp shells for

1984-1987 are reported as Brachiopoda in Fig.

3A. Data collected during 1984-1987 indicates

that brachiopods and acorn worms were scarce

in cores collected early on 1984 (dry season -

early rainy season) and increased to a maximum

towards the end of 1984 (late rainy - early dry

seasons). Sampling started shortly after the end

of the period of high temperatures produced

by the severe 1982-1983 ENSO. The impact

of this warming event on the Peruvian benthos

was described by Arntz, Brey, Tarazona, and

Robles (1987). In spite of the fact that sampling

effort was less after February 1985, numbers of

brachiopods and acorn worms were low and

patchy from early 1985 to early 1986, a period

when red tides were frequent in the upper Gulf

of Nicoya. The impact of these red tides on

other infaunal organisms such as the polychaete

worms has been discussed by Vargas-Zamora,

Sibaja-Cordero, Dean, and Solano-Ulate (2015).

Thus, we cannot rule out the possibility that the

abundances of brachiopods and acorn worms

found early on 1984, and those found during

1985-1986 were in some way also influenced

by the end of ENSO and red tides, respectively.

In summary, our data provides evidence of the

presence of acorn worms in the upper estuary

in sand-mud sediments.

The relatively low abundances of S. nudus

and Glottidia spp. and enteropneusts at the

Punta Morales sand mud flat particularly after

the red tides of 1985 may indicate that other

environmental factors were acting at this time.

In this context the 2013 survey data indicates

that sediments of the sand-mud flat changed to

finer, softer, and higher organic matter content

than those found in 1984, perhaps leading to the

lack of brachiopods and S. nudus in the samples.

Brachiopods and S. nudus seem to prefer coars-

er sediments like those found at Cocorocas in

2015. The enteropneusts appear more tolerant

to these changes since numbers near 10 ind./14

cores were found both in 1984 and 2013. Our

data also provides information on the relative

importance of these three groups in compari-

son with other main macro-invertebrates from

the Punta Morales flat collected during the

1984-1987 survey, like cephalochordates (Var-

gas & Dean, 2010), mollusks (Vargas-Zamora

& Sibaja-Cordero, 2011), echinoderms (Vargas

& Solano, 2011), crustaceans (Vargas-Zamora,

Sibaja-Cordero, & Vargas-Castillo, 2012) and

polychaetes (Vargas-Zamora, Sibaja-Cordero,

Dean, & Solano-Ulate, 2015).

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The presence of trace elements in brachio-

pod shells was reported by Jope (1965) and

included Fe, Mg, Mn and Cu. However, data

on the composition of shells and tissues of lin-

gulide brachiopods are scarce. Therefore, the

reported concentrations of metals in tissues and

shells of G. audebarti are the first informed for

this species. An important observation is that

concentrations were different in shells, pedi-

cles, and soft parts indicating different rates of

accumulation. Published reports on metal con-

centrations in other living brachiopods are rare.

The study by De Moreno, Gerpe, Moreno,

and Vodopivez (1997) from a pristine site in

Antarctica is a notable exception. They found

mean concentrations (μg/g dw) of Cd (4.33),

Cu (4.87) and Zn (26.70) in tissues of unidenti-

fied brachiopods. As a comparison, mean con-

centrations (μg/g dw) of these elements found

in soft parts of G. audebarti from the Cocoro-

cas sand flat were: Cd (5.20), Cu (12.88) and

Zn (55.20). Thus, concentrations of Cu and Zn

in G. audebarti from the tropical estuarine flat

were slightly higher than those from the Ant-

arctic pristine site.

Reports on the evaluation of trace metal

concentrations in tissues of sipunculans are

also scarce. De Moreno, Gerpe, Moreno and

Vodopivez (1997) found in unidentified whole

sipunculans from pristine sites in Antarctic

mean concentrations (μg/g dw) of Cd (0.47),

Cu (14.64) and Zn (69.9). Mean concentra-

tions (μg/g dw) of these metals found in whole

non-depurated S. nudus from the Cocorocas

sand flat during the 1996 survey were: Cd

(1.2), Cu (26) and Zn (56), and during the

2000 (Table 3) survey the concentrations were:

Cd (0.6), Cu (20.7) and Zn (81). Thus, mean

concentrations of these three metals in non-

depurated Antarctic and tropical sipunculans

were of the same orders of magnitude. Yan

and Wang (2002) found that sediments are

a direct source of metal accumulation in S.

nudus from a bay in China. They considered

that sediment concentrations of less than 0.9

μg/g of Cd, less than 49 μg/g of Cr, and less

than 140 μg/g of Zn represented a clean site,

and maximum concentrations (μg/g) were 9.0

for Cd, 61.6 for Cr and 428 for Zn at the con-

taminated sites. For comparison, maximum

mean concentrations (μg/g dw) of Cd, Cr, and

Zn in the Cocorocas flat sediments (1996 and

2000 surveys) were 1.1, 31.5, and 63, respec-

tively. Thus, the Cocorocas sediments may be

considered as relatively clean based on Yan and

Wang (2002) criteria. Moreover, Vargas, Acuña

González, Gómez, and Molina (2015) concluded

that metal concentrations in sediments from the

Cocorocas sand flat were those expected for a

region of the estuary not as yet impacted by

industrial activities. Wang, Yan, and Fan (2002)

found that S. nudus from the same bay in China

retained ingested sediment for about a day and

were able to egest unassimilated metals (Cd,

Cr, Zn) for up to 72 hours. Thus, the results

reported here are in agreement with those

found in China in specimens of S. nudus as

concentrations of metals (Fe, Mn, Ni, Zn, Pb)

in 72 hour depurated S. nudus from Cocoro-

cas were lower than those in non-depurated

organisms. The fraction of non-assimilated

sediments is important enough to influence

weight differences between depurated and non-

depurated S. nudus. Therefore, data on metal

concentrations in non-depurated S. nudus must

be viewed with caution. Ha, Nhuan, Ngoc and

Dung (2007) found in non-depurated S. nudus

from Vietnam mean ppm concentrations (μg/g

dw) of: Fe (441), Mn (3.7), Ni (4.7), Zn

(21.9), Pb (5.5), Cu (1.8), and Cd (0.3). With

the exceptions of Fe, the Vietnamese values

were of the same order of magnitude than

mean values reported here (μg/g dw) for Ni

(10.4), Zn (81), Pb (2.8) and Cd (0.63) found

in non-depurated S. nudus from Cocorocas.

Higher concentrations of Fe, Mn and Cu in

non-depurated S. nudus from Cocorocas, was

probably related to their abundance in ingested

and unassimilated sediments. Other species of

intertidal invertebrates, like certain filter feed-

ing bivalves, are able to lower their metal con-

centrations after depuration in clean sea water

as found by Vargas, Acuña González, Gómez,

and Molina (2015) for Fe, Mn, Ni, and Zn in

the razor clam Tagelus affinis from the Cocoro-

cas sand flat. Concentrations of Pb around

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20 μg/g dw in sediments, 9.3 μg/g dw in non-

depurated S. nudus, and 21 μg/g dw in shells

of G. audebarti collected during 1996 may be

related to the use of leaded fuel in outboard

engines operated by the artisanal fishing fleet at

the time; a small fishing village is located at the

mouth of the Lagarto River. However, Pb was

found in lower concentrations during the 2000

survey than during 1996. Other metals may

have been carried to the sand flat by this river,

which drains agricultural lands and brings

heavy loads of fine and coarse sediments dur-

ing the rainy season. Other compounds are also

transported by the Lagarto river. For instance,

a concentration of 41.4 ng/g dw of PCB was

found by Spongberg (2006) in S. nudus from

Cocorocas. However, this concentration was

relatively low when compared with PCB values

found in industrialized estuaries.

Data collected during the 2015 survey

confirmed the presence of the brachiopods

G. audebarti and G. albida at the Cocorocas

sand flat after both species were first collected

there by Emig and Vargas (1990). The survey

yielded a maximum shell length of 26 mm for

G. albida, and 38 mm for G. audebarti, which

are in agreement with measurements made 25

years ago. Jones and Barnard (1963) conducted

a spatial survey of the abundance of G. albida

in subtidal coastal sediments of the California

(33

N) coast. This species was more common

at depths from 22 m to 47 m on a wide variety

of sediments. The size distribution of 8 976

specimens of G. albida collected by Jones and

Barnard (1963) was dominated (86 %) by indi-

viduals of less than 5 mm, and only 1 % includ-

ed individuals of 21-50 mm long. In tidal flats of

the Colorado River mouth (31

N) in the Gulf of

California, small individuals of G. palmeri were

rare and this species was found in patches of

individuals of mean shell length of about 38 mm

(Kowalewski, 1996).

On the other hand, at Cocorocas indi-

viduals smaller than 15 mm long characterized

G. albida. This lingulide almost vanished from

the sand flat in December, 2015. In addi-

tion, as found by Jones and Barnard (1963),

G. albida survives well at its Northern range in

non-estuarine coastal waters cooler than 20

and in depths shallower than 60 m. In 1980 a

grab survey of 41 subtidal (6 to 65 m) stations

in the Gulf of Nicoya included 15 stations in the

upper estuary. However, as reported by Vargas,

Dean, Maurer and Orellana (1985) only one

Glottidia sp. specimen was found during the

survey (Ballena Bay, 20 m, lower estuary). Estu-

arine low salinities and higher intertidal tem-

peratures at the Cocorocas sand flat enhanced

by the ENSO 2015 (NOAA, 2016) conditions

may be among the possible factors preventing

this subtidal species to reach a relatively stable

presence at the intertidal flat. Moreover, tem-

peratures of 40

C and 41

C were recorded at

noon in tide pools from the Cocorocas sand flat

on July 6 and December 15, 2015, respectively.

The presence of G. audebarti in the Gulf

of Nicoya is at the middle of the latitudinal

range of this mainly intertidal species (Emig,

1997) and these two factors may influence its

relatively stable presence at Cocorocas. Paine

(1963) described the life cycle of G. pyramidata

living in intertidal sand bars on the West coast

of Florida (29

N) in salinities ranging from

19 psu to 35 psu. At these sand bars most of

G. pyramidata individuals live for about a year

with some individuals living up to 20 months.

However, comparative information is lacking on

the life span of both Glottidia species from the

Gulf of Nicoya.

Natural mortality must be important at

Cocorocas, although empty shells were never

observed. Paine (1963) also points out that the

phosphatic shell of Glottidia and its thin organic

periostracum decay rapidly in the sediments.

Predation on Glottidia spp. by sandpiper shore

birds has been observed by Pereira (1996) at

nearby sand flats in the Gulf of Nicoya. Naticid

snail boreholes are frequently found on mollus-

can shells at the Punta Morales region (Vargas-

Zamora & Sibaja-Cordero, 2011), but no such

boreholes or traces of them have been observed

on brachiopod shells from the sites.

At the Cocorocas sand flat maximum

mean densities of G. audebarti and G. albida

were 8.6 and 18.2 ind./m

, respectively. Jones

and Barnard (1963) reported densities of

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G. albida of 20 ind./m

in coarser sediments

where the onuphid polychate Diopatra sp.

was common, while higher densities (up to

500 ind./m

) coincided with the presence of the

ophiuroid Amphioplus hexacanthus. At both

the Cocorocas and Punta Morales flats the

infaunal ophiuroid, Amphipholis geminata and

the onuphid Diopatra ornata are conspicuos

elements of the macrofauna (Ditmann & Var-

gas, 2001; Vargas & Solano, 2011). In sand

flats from Australia (19

S) L. anatina reached

densities of 864 ind./m

(Kenchingtom & Ham-

mond, 1978), while in intertidal flats in the Gulf

of California (31

N) G. palmeri had densities

of up to 300 ind./m

(Kowalewski, 1996). A

drastic decline in abundance, from a maximum

of 500 ind./m

(mean 25-50 ind./m

) in 1969

to 0.94 ind./m

in 2007, has been reported for

L. reevii in reef flats from Hawaii (21

N). The

decline was related to a decrease in organic mat-

ter input and algal mats covering the sediments

(Hunter, Krane, & Fitzpatrick, 2008). At the Bay

of Bengal (India, 14

N) L. translucida reached

densities between 4 ind./m

and 92 ind./m

(mean

46 ind./m

) estimated from 0.25 m

quadrats col-

lected along three transects on an intertidal beach

under the influence of a brackish water river

(Raughnathan & Jothinayagam, 2007). Thus,

densities of both G. audebarti and G. albida at

Cocorocas appear relatively modest when com-

pared with data from higher latitudes, but similar

to those of G. translucida found in a tropical

habitat in India under the influence of a river.

The 2015 sampling at the Cocorocas sand

flat also confirmed the presence of the sipun-

culan S. nudus. Individuals representing nine

weight groups of this peanut worm were found,

but with most of the individuals were in the 0.1

to 1.0 g range. Representatives of this range

increased in abundance from July to December

indicating possible growth of the individuals.

Both new (lighter) and heavier (probably older)

specimens were present during the sampling

period, which may represent a relatively stable

population. A density of 5 ind./m

of S. nudus

is mentioned by Saiz-Salinas (1993). There-

fore, densities of about 34.5 ind./m

found at

the Cococorcas sand flat are relatively high.

However, their mean size of 45 mm is relatively

small if the maximum size (340 mm) also cited

by Saiz-Salinas (1993) and a photograph in Ha,

Nhuan, Ngoc, and Dung (2007) are considered

for comparison. These latter authors indicate

that S. nudus is more abundant in sand flats

from Vietnam where sediments contain more

than 80 % sand, and is rarely found if the

sand content is less than 60 %. These values

may explain the relatively high abundance of

S. nudus in Cocorocas sediments (90 % sand)

when compared to their low abundance in the

65 % sand content of the Punta Morales flat.

The distribution of individuals in space

is a fundamental characteristic of soft-bottom

species (Thrush, 1991). G. palmeri and other

lingulides occur in patches in the intertidal

zone (Kowalewski, 1996). Our data indicates

that spatial patchiness characterizes the abun-

dance of the three species at the Cocorocas

sand flat. The two brachiopods and S. nudus

were frequently found together within a single

quadrat area, as illustrated for G. audebarti

and S. nudus for the 18 quadrats collected on

Dec. 15, 2015. The number of individuals col-

lected in a single quadrat ranged from 0 to 11

(G. audebarti), 0 to 18 (G. albida) and 0 to 20

(S. nudus), which may indicate subtle differ-

ences in sediment grain size and other factors

(like presence of adults) attractive or dissuasive

for site selection by settling larvae and later

survival of juveniles (Gray & Elliot, 2009).

Spatial and temporal patchiness is also

important in the context of fishing potential

of certain species since relatively high mean

densities at a given sediment spot or time may

be misleading. The sipunculan S. nudus is not

as yet exploited commercially in Costa Rica,

but it has been used as fish bait for genera-

tions. The peanut worm coexists at Cocorocas

with the lamp shells and the small razor clam

T. affinis which is already under moderate

fishing pressure.

Brachiopods of the genus Lingula are har-

vested commercially in Southeast Asia (Brusca,

Moore, & Shuster, 2016). Future exploitation

of populations of lamp shells in the Gulf of

Nicoya may be expected, and management

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policies are urgently needed to prevent future

overfishing of these species. Other tidal flat

species from the Gulf of Nicoya, like Anadara

spp. ark clams (Stern-Pirlot & Wolff, 2006)

and the giant polychaete Americonuphis reesei

(Rojas & Vargas, 2008) are examples of living

resources where management strategies have

been implemented with relatively good results

in this tropical estuary.

ACKNOWLEDGMENTS

We thank Davis Morera and Eleazar Ruiz

for their help in the field collections during

2000, 2013 and 2015. Eddy Gómez and Johan

Molina helped with the chemical laboratory

work. We thank three anonymous reviewers for

comments on the manuscript. Data collected

during 1984-1987 was part of the Ph. D. Dis-

sertation by the senior author at the University

of Rhode Island, U.S.A. We thank the late John

S. Gray for supporting the 1996 trace metal

analysis at the University of Oslo, Norway.

Trace metal anaylsis during 2000 were made

possible by a grant from the Costa Rica-Unit-

ed States of America Foundation (CR-USA)

to the senior author (Project VI-808-A0-506

Coastal pollution in Costa Rica). This paper

was prepared as part of projects VI-808-B3-113

The benthos of Punta Morales-30 years later,

and VI-808-B3-127 Microbial ecology and

marine biogeochemistry of the Gulf of Nicoya,

funded by research grants from the University

of Costa Rica.

RESUMEN

Braquiópodos, sipuncúlidos, enteropneustos y

metales en dos planicies estuarinas de marea, Pacífico,

Costa Rica. Son raros los reportes sobre las abundancias

y concentraciones de metales en invertebrados estuarinos

de la zona de entre-mareas del Pacífico Este Tropical. Los

objetivos de este informe son el hacer accesibles datos sobre

las abundancias (1984-1987, 49 fechas; 2013, 12 fechas) de

sipuncúlidos, braquiópodos y hemicordados en una planicie

arenoso-fangosa y sobre metales traza (1996, 2000) y abun-

dancias (2015, 3 fechas) de sipuncúlidos y braquiópodos

en una planicie arenosa en el estuario del Golfo de Nicoya

(10

N-85

W). Barrenos (17.7 cm

) fueron recolectados

en la planicie arenoso-fangosa y cuadrantes (0.2 m

) en la

arenosa. Las planicies contrastaron en sus contenidos de

arena (65 % vs 90 %) y de limo + arcilla (31.5 % vs 5.6 %).

En la planicie arenoso-fangosa (1984-87: 1.83 m

) los

sipuncúlidos estuvieron representados por 13 individuos, los

braquiópodos por 129 y los hemicordados enteropneustos

por 185, con densidades estimadas de: 5.7, 29, y 40 ind. /m

respectivamente. Análisis de metales traza (Fe, Mn, Ni, Cr,

Cd, Zn, y Pb) por Espectrometría de Absorción Atómica

(AAS) fueron hechos en especímenes de Sipunculus nudus

(Sipuncula) y Glottidia audebarti (Brachiopoda). Con-

centraciones máximas promedio en S. nudus fueron: para

gusanos no-depurados, Fe (16.0 mg/g dw) > Mn (165 µg/g

dw) > Zn (81 µg/g dw) > Cu (26 µg/g dw) > Cr (11 µg/g

dw) > Ni (10.4 µg/g dw) > Pb (9.3 µg/g dw) > Cd (1.2 µg/g

dw). Para gusanos depurados por 72 horas: Fe (5.0 mg/g

dw) > Mn (61 µg/g dw) > Zn (39 µg/g dw) > Cu (24 µg/g

dw) > Ni (8.4 µg/g dw) > Pb (2.7 µg/g dw) > Cd (0.62 µg/g

dw). Para G. audebarti: Fe (1.6 mg/g dw-partes suaves)

> Zn (123.5 µg/g dw-partes suaves) > Cu (31.4 µg/g dw-

pedículos) > Pb (21.0 µg/g dw-conchas) > Cd (5.2 µg/g

dw-partes suaves) > Cr (4.7 µg/g dw-conchas). Para sedi-

mentos; Fe (46 mg/g dw) > Mn (41.3 µg/g dw) > Zn (63

µg/g dw) > Cu (36.2 µg/g dw) > Cr (31.5 µg/g dw) > Pb

(21.1 µg/g dw) > Ni (16.1 µg/g dw) > Cd (1.1 µg/g dw).

Estas concentraciones fueron esperables para un estuario

no industrializado. En la planicie arenosa (Area muestreada:

10.6 m

) 76 individuos de G. audebarti, 112 de G. albida y

366 de S. nudus fueron recolectados en el 2015, con densida-

des estimadas de: 7.1, 10.5, y 31 ind. /m

, respectivamente.

Densidades de G. audebarti y G. albida fueron relativamen-

te bajas, mientras que las de S. nudus fueron relativamente

altas cuando se les comparó con otros reportes. La longitud

de la concha de G. audebarti varió entre 9.0 mm y 38.0 mm

y entre 6.0 mm a 29.0 mm la de G. albida. Estos ámbitos

estuvieron dentro de los encontrados para estos lingúlidos

en otros sitios. La longitud promedio de S. nudus fue 41 mm

y el peso máximo fue de 1.6 g que son pequeños. En la

planicie arenoso-fangosa no se encontró braquiópodos en el

2013, ni enteropneustos en la planicie arenosa en el 2015.

G. audebarti tuvo una presencia relativamente estable,

mientras que G. albida casi desapareció de las muestras al

final del 2015. La distribución espacial de las tres especies

fue de tipo agregado en ambas planicies. Fuertes eventos

ENSO durante 1983 y 2015, así como mareas rojas en 1985,

pueden haber influenciado las abundancias.

Palabras clave: Glottidia, Sipunculus, Apionsoma, gusanos

bellota, bentos tropical, infauna, contaminación, depuración.

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